CN117881788A - Expression vectors, bacterial sequence-free vectors, and methods of making and using the same - Google Patents

Expression vectors, bacterial sequence-free vectors, and methods of making and using the same Download PDF

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CN117881788A
CN117881788A CN202280055798.2A CN202280055798A CN117881788A CN 117881788 A CN117881788 A CN 117881788A CN 202280055798 A CN202280055798 A CN 202280055798A CN 117881788 A CN117881788 A CN 117881788A
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sequence
seq
vector
nucleic acid
aspects
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R·斯拉夫切夫
N·纳菲西
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Medical Phage Biopharmaceutical Co
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Medical Phage Biopharmaceutical Co
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Priority claimed from PCT/IB2022/055620 external-priority patent/WO2022264095A1/en
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Abstract

The present disclosure provides expression vectors, bacterial sequence-free vectors, such as ministring DNA (msDNA), and methods of making bacterial sequence-free vectors, including use of vector production systems. The invention also provides compositions comprising the carrier, and uses of the carrier and compositions.

Description

Expression vectors, bacterial sequence-free vectors, and methods of making and using the same
Sequence Listing with references submitted electronically through EFS-Web
The contents of the electronically submitted sequence listing (name: 4471_007003_seqlisting_st25.txt; size: 216,898 bytes; date of creation: 2022, 6, 15 days) are incorporated herein by reference in their entirety.
Technical Field
The present disclosure provides expression vectors, bacterial sequence-free vectors, vector production systems for preparing the bacterial sequence-free vectors, and uses thereof.
Background
Gene therapy has significant therapeutic prospects, but challenges remain in achieving its potential.
Most clinical trials employ viral delivery systems such as adenovirus vectors, lentiviral vectors, and adeno-associated virus vectors. Although viral systems have evolved, they have altered transduction and transgene expression efficiencies and there is still concern about adverse effects such as inflammation and immune responses or insertional mutagenesis. Furthermore, production, purification and storage of viral vectors is often expensive, highly variable and inefficient. See, e.g., lingelbach, d., drug Development & Delivery 20 (5): 50-54 (2020); wright, J.F., gene Therapy 15:840-848 (2008).
Non-viral vectors have also been studied as gene therapy delivery systems. Although safer than viral vectors, the effectiveness of non-viral vectors can be limited due to, for example, low transgene expression levels and expression persistence. See, for example, kay, m., nature Reviews Genetics 12:316-328 (2011).
Thus, there is a need for improved vectors, such as the improved vectors described herein.
Summary of The Invention
The present invention relates to an expression vector comprising: (a) a backbone sequence; (b) comprises the sequence: (i) an expression cassette comprising a nucleic acid sequence of interest, (ii) a first target sequence for a first recombinase located 5 'to the expression cassette, (iii) a second target sequence for the first recombinase located 3' to the expression cassette, and (iv) one or more further target sequences for one or more further recombinases, wherein the further target sequences are integrated within the first and second target sequences in non-binding regions of the first recombinase: and (c) one or more of the following: (i) an endonuclease target sequence integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the endonuclease target sequence is located between the backbone sequence and the cleavage site of the first recombinase and the one or more other recombinases, (ii) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12, wherein the synthetic enhancer is integrated between the 3 'end of the first target sequence of the first recombinase and the 5' end of the other enhancer or promoter in the expression cassette, (iii) a Cytomegalovirus (CMV) enhancer integrated between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter in the expression cassette, (iv) a 5 'non-translated region (5' UTR) integrated between the promoter and the nucleic acid sequence of interest in the expression cassette, (vii) a signaling element located between the polynucleotide of interest and the polynucleotide host cell (sense gene) in the expression cassette, (vii) a signaling element located between the polynucleotide of interest (sense gene) and the polynucleotide cassette (sense gene) and the polynucleotide of interest (sense gene) and (sense gene) a polynucleotide (sense gene) located between the polynucleotide cassette (sense gene) and the host cell (sense gene) of interest (sense gene) and a polynucleotide (sense gene) located between the polynucleotide (sense gene) and the sense gene (sense gene) of the sense gene (sense gene) and the sense gene (sense gene) or a polynucleotide (sense gene) located between the polynucleotide (sense gene) and the sense gene, or (viii) a DNA core targeting sequence (DTS) integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the DTS is located between the expression cassette and the cleavage sites of the first recombinase and the one or more other recombinases.
In some aspects, the expression vector comprises an endonuclease target sequence integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the endonuclease target sequence is located between the backbone sequence and the cleavage site of the first recombinase and the one or more other recombinases. In some aspects, the endonuclease target sequence is integrated within the first and second target sequences of the first recombinase. In some aspects, the endonuclease target sequence is used to home an endonuclease (homing endonuclease). In some aspects, the endonuclease target sequence is an endonuclease target sequence of I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-Dmo I, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfeuI, PI-PkoII, I-PoI, I-PpoI, PI-PspI, I-ScaI, I-SceI, I-SecIII, I-SceIV, I-SceVI, I-SceI, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, I-Tsp061I, or I-Tsi141I. In some aspects, the endonuclease target sequence is for I-SceI. In some aspects, the endonuclease target sequence is for PI-SceI. In some aspects, the endonuclease target sequence is for a Cas endonuclease. In some aspects, the Cas endonuclease is Cas9.
In some aspects, the expression vector comprises a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 and integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of another enhancer or promoter. In some aspects, the synthetic enhancer comprises multiple contiguous copies of the sequence set forth in SEQ ID NO:12, at least about 90% identical. In some aspects, the synthetic enhancer comprises a nucleotide sequence that hybridizes to SEQ ID NO:46, and a nucleic acid sequence having at least about 90% identity. In some aspects, the synthetic enhancer is integrated 5' to the chicken β -actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 47 is integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a CMV enhancer integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 or SEQ ID NO. 46. In some aspects, the CMV promoter is integrated 3 'of the CMV enhancer and 5' of the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 or SEQ ID NO. 39, said nucleic acid sequence being integrated between the first target sequence of the first recombinase and the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a 5'utr comprising an intron, wherein the 5' utr is integrated in the expression cassette between the promoter and the nucleic acid sequence of interest. In some aspects, the intron comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, and a nucleic acid sequence that is at least about 90% identical. In some aspects, the 5' utr further comprises a non-coding sequence integrated in the intron. In some aspects, the 5' UTR comprises a non-coding sequence between two nucleotides integrated in the intron, wherein the two nucleotides correspond to any two nucleotides from position 25 to 55 of SEQ ID NO. 1. In some aspects, the non-coding sequence is an S/MAR. In some aspects, the S/MAR is MAR-5. In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:3, at least about 90% identical. In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:5, and a nucleic acid sequence at least about 90% identical. In some aspects, the promoter is a chicken β -actin promoter. In some aspects, the promoter is a CMV promoter. In some aspects, the promoter is integrated 3' to the CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 or SEQ ID NO. 46.
In some aspects, the expression vector comprises a polyadenylation signal integrated at the 3' end of the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the expression vector comprises a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the vertebrate chromatin insulator is a 5' -HS4 chicken β -globin insulator (cHS 4). In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the expression vector comprises a WPRE integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the expression vector comprises an S/MAR integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the S/MAR is MAR-5. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the expression vector comprises an enhancer sequence flanking each of the first and second target sequences of the first recombinase. In some aspects, the expression vector comprises at least two enhancer sequences flanking each side of the first and second target sequences of the first recombinase. In some aspects, the enhancer sequence is an SV40 enhancer sequence.
In some aspects, the expression vector comprises a DTS integrated within the first and/or second target sequences of the first recombinase and in the non-binding region of the first recombinase and the one or more other recombinases, wherein the DTS is located between the expression cassette and the cleavage site of the first recombinase and the one or more other recombinases. In some aspects, the DTS is an SV40 enhancer sequence. In some aspects, the DTS is cell-specific.
In some aspects, the first and second target sequences and the one or more additional target sequences are selected from the group consisting of PY54 pal site, N15telRL site, loxP site, and,telRL site, FRT site, phiC31 attP site and lambda attP site. In some aspects, the expression vector comprises all of these target sequences. In some aspects, the expression vector comprises a pal site and telRL, loxP, and FRT recombinase target binding sequences integrated within the pal site. In some aspects, the first and second target sequences of the first recombinase each comprise the nucleic acid sequence of SEQ ID NO. 33.
In some aspects, the expression vector is used to produce a bacterial sequence-free vector. In some aspects, the bacterial sequence-free vector is a circular covalently closed vector. In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
The present disclosure relates to a vector production system comprising a recombinant cell encoding a recombinase under the control of an inducible promoter, wherein the recombinant cell comprises any of the above-described expression vectors, and wherein the recombinase targets one of the first and second target sequences of the first recombinase or one or more other target sequences of one or more other recombinases in the expression vector. In some aspects, the recombinase is TelN, tel, cre or Flp.
In some aspects, the recombinant cell further encodes an endonuclease under the control of an inducible promoter, wherein the endonuclease targets an endonuclease target sequence in an expression vector comprising the endonuclease target sequence. In some aspects, the endonuclease is a homing endonuclease. In some aspects, the homing endonuclease is I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-Dmo I, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PpfuI, PI-PkoII, I-PoI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SecIII, I-SceIV, I-SceVI, I-SceI, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, I-Tsp061I or I-Tsi141I. In some aspects, the endonuclease is I-SceI. In some aspects, the endonuclease is PI-SceI. In some aspects, the recombinant cell encodes a nuclease genome editing system comprising the endonuclease. In some aspects, the nuclease genome editing system is a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) nuclease system comprising a guide RNA and a Cas endonuclease. In some aspects, the Cas endonuclease is Cas9. In some aspects, the inducible promoter is thermally regulated, chemically regulated, IPTG regulated, glucose regulated, arabinose-inducible, T7 polymerase regulated, cold shock inducible (cold-shock inducible), pH inducible, or a combination thereof.
The present disclosure relates to a method of producing a bacterial sequence-free vector comprising incubating any of the above vector production systems under conditions suitable for expression of a recombinase. In some aspects, the method further comprises incubating any of the above endonuclease-encoding vector production systems under conditions suitable for expression of the endonuclease. In some aspects, the method further comprises incubating any of the above-described vector production systems encoding a nuclease genome editing system under conditions suitable for expression of the nuclease genome editing system. In some aspects, the method further comprises harvesting the bacterial sequence-free vector.
The present disclosure relates to a bacterial sequence-free vector produced by any of the above methods of producing a bacterial sequence-free vector.
The present disclosure relates to a bacterial sequence-free vector comprising: (a) An expression cassette comprising a nucleic acid sequence of interest, and (b) one or more of: (i) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 located 5 'to another enhancer or promoter in the expression cassette, (ii) a CMV enhancer located 5' to the promoter in the expression cassette, (iii) a 5'UTR comprising an intron located between the promoter and the nucleic acid sequence of interest integrated in the expression cassette, (iv) a vertebrate chromatin insulator located between the nucleic acid of interest and the polyadenylation signal integrated in the expression cassette, (v) a WPRE located between the nucleic acid of interest and the polyadenylation signal integrated in the expression cassette, (vi) an S/MAR located between the nucleic acid of interest and the polyadenylation signal integrated in the expression cassette, or (vii) a DTS located 5' to the expression cassette.
In some aspects, the bacterial sequence-free vector comprises a synthetic enhancer 5' to another enhancer or promoter in the expression cassette, the synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12. In some aspects, the synthesis enhancer comprises multiple consecutive copies of a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 12. In some aspects, the synthetic enhancer comprises a nucleotide sequence that hybridizes to SEQ ID NO:46, and a nucleic acid sequence having at least about 90% identity. In some aspects, the synthesis enhancer is integrated 5' to the chicken β -actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 47 is integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a CMV enhancer 5' to the promoter in the expression cassette. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 or SEQ ID NO. 46. In some aspects, the CMV promoter is integrated 3 'of the CMV enhancer and 5' of the nucleic acid sequence of interest.
In some aspects, the bacterial-free sequence vector comprises a nucleic acid sequence 5' to the nucleic acid sequence of interest and a sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39, and a nucleic acid sequence having at least about 90% identity.
In some aspects, the bacterial-free sequence vector comprises a 5' utr comprising an intron located between the promoter and the nucleic acid sequence of interest integrated in the expression cassette. In some aspects, the intron comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, and a nucleic acid sequence that is at least about 90% identical. In some aspects, the 5' utr further comprises a non-coding sequence integrated in the intron. In some aspects, the 5' UTR further comprises a non-coding sequence integrated between two nucleotides in the intron, wherein the two nucleotides correspond to any two nucleotides from 25 to 55 of nucleotide positions of SEQ ID NO. 1. In some aspects, the non-coding sequence is an S/MAR. In some aspects, the S/MAR is MAR-5. In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:3, at least about 90% identical. In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:5, and a nucleic acid sequence at least about 90% identical. In some aspects, the promoter is a chicken β -actin promoter. In some aspects, the promoter is a CMV promoter. In some aspects, the promoter is integrated 3' to the CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 or SEQ ID NO. 46.
In some aspects, the bacterial sequence-free vector comprises a polyadenylation signal integrated 3' to the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the bacterial-free sequence vector comprises a vertebrate chromatin insulator integrated in an expression cassette between a nucleic acid of interest and a polyadenylation signal. In some aspects, the vertebrate chromatin insulator is cHS4. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the bacterial-free sequence vector comprises a WPRE integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.
In some aspects, the bacterial-free sequence vector comprises an S/MAR integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the S/MAR is MAR-5.
In some aspects, the bacterial sequence-free vector comprises enhancer sequences flanking each side of the expression cassette. In some aspects, the bacterial sequence-free vector comprises at least two enhancer sequences flanking each side of the expression cassette. In some aspects, the enhancer sequence is an SV40 enhancer sequence.
In some aspects, the bacterial sequence-free vector comprises a DTS located 5' to the expression cassette. In some aspects, the DTS is an SV40 enhancer sequence. In some aspects, the DTS is cell specific.
In some aspects, the bacterial sequence-free vector is a circular covalently closed vector.
In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
The present disclosure relates to recombinant cells comprising any of the above-described expression vectors or any of the above-described bacterial sequence-free vectors.
The present disclosure relates to a composition comprising any of the above expression vectors or any of the above bacterial sequence-free vectors. In some aspects, the composition further comprises a delivery agent. In some aspects, the delivery agent is a nanoparticle. In some aspects, the delivery agent comprises a targeting ligand. In some aspects, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
The present disclosure relates to methods of treating a disease or disorder in an individual in need thereof, comprising administering to the individual any of the above expression vectors, any of the above bacterial sequence-free vectors, or the above pharmaceutical compositions.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 1.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 2.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 3.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 5.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 46.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 13.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 14.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 15.
In some aspects, any of the above polynucleotides comprising a nucleic acid sequence at least about 90% identical to any of SEQ ID NOs 13-15 further comprises 100-120 adenine nucleotides at the 3' end of the nucleic acid sequence.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 16.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 17.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 18.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 35.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 36.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 37.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 38.
The present disclosure relates to polynucleotides comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 39.
The present disclosure relates to an expression vector comprising any of the polynucleotides described above.
The present disclosure relates to an expression vector comprising: a polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to any one of SEQ ID nos. 2, 3 or 5; and (i) a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOS.13-18, or (ii) a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOS.13-15 and 100-120 adenine nucleotides at the 3' end of said nucleic acid sequence.
The present disclosure relates to a method of gene editing comprising inserting a nucleic acid sequence of interest from any of the above-described expression vectors, any bacterial-free sequence vectors, or any of the above-described pharmaceutical compositions into a target site for gene editing. In some aspects, gene editing is performed by non-homologous end joining. In some aspects, gene editing is performed by homology-directed repair (homology-directed repair).
Brief Description of Drawings
FIG. 1 shows a vector map of the expression vector pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS.
FIG. 2 shows a vector map of the expression vector pcDNA-CMV-5' UTR-secNLuc-P2A-eGFP-bGHPA.
FIG. 3 shows a micrograph of fluorescence in HEK-293 cells assessed by live imaging (live imaging). (a) shows negative control cells exposed to plasmid-free lipofectamine, (B) shows cells transfected with the expression vector of fig. 1, (C) shows cells transfected with the expression vector of fig. 2, (D) shows positive control cells transfected with the parental expression vector pGL 2-SS-CAG-eGFP-BGpA-SS (PP-CAG-GFP), wherein eGFP is expressed under the control of the CAG promoter.
FIG. 4 shows a bar graph of the relative fluorescence intensities of cells transfected according to FIGS. 3 (A) - (D). "pGL2-SecNLuc-eGFP" in FIGS. 4-5 represents cells transfected with pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS expression vector of FIG. 1. "pcDNA-SecNLuc-eGFP" in FIGS. 4-5 represents cells transfected with the pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA expression vector of FIG. 2.
FIG. 5 shows a bar graph of relative luciferase intensities in culture medium of cells transfected according to FIGS. 3 (A) - (C).
FIG. 6 shows a vector map of the expression vector pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS.
FIG. 7 shows a vector map of the expression vector pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS.
FIG. 8 shows a vector map of the expression vector pGL 2-SS-CMV-UTR 2-SecNLuc-2A-eGFP-WPRE-BGpA-SS.
FIG. 9 shows a line graph of long-term luciferase activity in HEK-293 cell medium at days 2, 6, 10, 14, 17, 20, 27 and 34 after electroporation of cells with the expression vectors of FIG. 1 (pGL 2-SecNLuc-eGFP), FIG. 6 (WPRE), FIG. 7 (5 'UTR1+WPRE) and FIG. 8 (5' UTR2+WPRE) as compared to a negative control, wherein the luciferase activity is indicated by luminescence expressed in relative luminometer units (also referred to as relative luminometer units, RLU), wherein the negative control is a cell electroporated with puc57 plasmid without a mammalian expression cassette (negative control (no plasmid)). * P <0.05, =p <0.01, =p <0.001, and=p <0.0001.
FIG. 10 shows bar graphs of luciferase activity in culture medium at passages 1, 2, 3 and 5 of transfected and negative control HEK-293 cells as described in FIG. 9, wherein the enzyme activity is indicated by RLU luminescence.
FIG. 11 shows a plot of relative luciferase intensities in HEK-293 cells at 1, 2, 3, 4, 5, 6 and 7 passages (corresponding to days 8, 15, 24, 31, 38, 45 and 52, respectively) after electroporation of cells with the expression vector of FIG. 7 (2 nd gen pDNA (CMV+U1+W)), msDNA generated from the expression vector of FIG. 7 (2 nd gen msDNA (CMV+U1+W)), or the expression vector containing a luciferase transgene of FIG. 2 (conventional pcDNA). In comparison to the conventional pcDNA dataset, p <0.01, p=p <0.001, and p <0.0001.
FIG. 12 shows fluorescence in cells transfected with the expression vectors of FIG. 1 or FIG. 7, as described in FIG. 9. (A) Microphotographs showing fluorescence in HEK-293 cells assessed by live cell imaging at passages 1, 2, 3 and 5 are shown. (B) A line graph showing eGFP positive (gfp+) cells observed in the field of view of three live cell fluorescence images per generation; insignificant (ns) =p >0.05. (C) The dot pattern of (c) shows the Mean Fluorescence Intensity (MFI) of gfp+ cells measured in three live cell fluorescence images of passage 5. The bar graph corresponding to each construct shows the average MFI value of all gfp+ cells measured. * P <0.0001.
FIG. 13 shows a plot of RLU/mg protein in plasma collected from wild-type mice on days 1, 3, 7, 10, 15, 22, 28, 42 and 56 after single hydrodynamic tail intravenous injection of 50 μg pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control, PSNLuc), pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS (pCAGLuc), or pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS (pGSNLuc-WPRE).
FIG. 14 shows a line graph of RLU/mg protein in plasma collected from wild-type mice on days 1, 3, 7, 10, 15, 22, 28, 42 and 56 after single hydrodynamic tail intravenous injection of 50 μg of pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control, PSNLuc), pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS (pCAGLuc), or pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS.
FIG. 15 shows a plot of RLU/mg protein in plasma collected from wild-type mice on days 1, 3, 7, 10, 15, 22, 28, 42 and 56 after single hydrodynamic tail intravenous injection of 5 μg of pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control, pDNA CMV-U (no SSeq)), pGL 2-SS-CAG-SecNLuc-2A-BGpA-SS (SSeq pDNA CAG-G) or pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS.
FIG. 16 shows a line graph of RLU/mg protein in plasma collected from wild-type mice on days 1, 3, 7, 10, 15, 22, 28, 42 and 56 after single hydrodynamic tail intravenous injection of 5 μg of pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control, no SSeq pDNA CMV-U), pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (2 XSseq pDNA CAG-W), or msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (2 XSseq msDNA CAG-W).
Figures 17A-D show bar graphs of GFP expression measured by ELISA in livers from wild-type mice at day 56 after a single hydrodynamic tail vein injection of 5 μg of the vector of figure 16 and negative control mice without vector injection. GFP concentrations (in μg/mL) are shown in (A). (B) Shown are μg GFP normalized to μg of total protein. (C) shows μg GFP normalized relative to total tissue g. (D) shows GFP expression levels relative to the control. * P <0.05, < P <0.01, < P <0.001, < P <0.0001.
FIG. 18 shows bar graphs of cytoplasmic GFP protein concentration (pg/mL) in the liver from wild type mice on day 56 after a single hydrodynamic tail vein injection of 5 μg of the vector of FIG. 16 and negative control mice without vector injection.
FIG. 19 shows a bar graph of total flux in photons/second from in vivo whole body bioluminescence imaging after a single intravenous tail vein injection of Lipid Nanoparticle (LNP) carrier (vehicle (control)) or following lipid complex (lipoplex) in mice, wherein the lipid complex is LNP and msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (LNP-2G msDNA-CAG-secretory NanoLuc), pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS (LNP-2G ppDNA-CAG-secretory NanoLuc), msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (LNP-2G msDNA-CMV-secretory NanoLuc), pGL 2-SS-SS-CMV-UTR 1-SecNLuc-2A-eGFP-BGpA-SS (LNP-2G ppDNA-secretory NanoLuc), or DNA-pcNL-P2A-eGFP-eGHpA-PdNA-conventional lipid complex, wherein the bar graph shows total flux from left to right for days 1, 3, 10, 30, 58, 92, 119, and 174 after injection for each injection. Bar graphs of LNP-2G msDNA-CAG-secretory NanoLuc injection are boxed in dashed lines.
FIG. 20 shows a micrograph of Green Fluorescent Protein (GFP) expression in sagittal brain sections from msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA injected mice cortex, thalamus, brainstem and cerebellum. White arrows indicate transgene expression. Nuclei were indicated by staining with diamidino-2-phenylindole (DAPI).
FIGS. 21-22 show micrographs of GFP expression in sagittal brain sections from mice injected with msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA, cortex and thalamus (FIG. 21) and cerebellum and brain stem (FIG. 22). Neurons are indicated with the neuron marker NeuN.
FIGS. 23-24 show micrographs of GFP expression in sagittal brain sections from mice injected with msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA, cortex and thalamus (FIG. 23) and cerebellum and brainstem (FIG. 24). Neurons are indicated with the neuron marker NeuN.
FIGS. 25-26 show bar graphs of luminescence associated with luciferase expression in human T cells (Pan-T (TA+) cells, FIG. 25) or hepatocytes (Huh 7 cells, FIG. 26) 3 days and 5 days after transfection with lipid complexes of lipid nanoparticle carrier (LNP) with pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control, LNP-normal pDNA-CMV-secretory NLuc), pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS (LNP-ppDNA-CMV-secretory NLuc), msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (LNP-msDNA-CMV-secretory NLuc), pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS (LNP-ppDNA-CAG-secretory NLuc) or msDNA-segp-WPRE-BGpA (LNP-dna-bg-secretory NLuc).
FIGS. 27A-C, 28A-C and 29A-B show Fluorescence Activated Cell Sorting (FACS) scatter plots of knock-in KI efficiency (Q3) of a gene of interest (GOI) 3 days (FIG. 27A-C), 7 days (FIG. 28A-C) and 15 days (FIG. 29A-B) after transfection of the CRISPR gene editing system and conventional plasmid or msDNA carrying GOI flanked by 5 'and 3' homology arms (HDR-GOI-HDR). Fig. 27 and 28 (a) show FACS scatter plots of control wild-type (WT) -induced pluripotent stem cells (ipscs) of any HDR KI without GOI. Fig. 27-28 (B) and fig. 29 (a) show FACS scatter plots of the HDR KI post iPSC for GOI using conventional plasmids (plasmid DNA HDR-GOI-HDR). Fig. 27-28 (C) and fig. 29 (B) show FACS scatter plots of post-HDR KI ipscs for GOI using msDNA (msDNA HDR-GOI-HDR).
FIG. 30 shows a vector map of expression vector SS-CMV-UTR 1-SecNLuc-2A-eGFP-3' UTR [2hBGpA-A120] -SS.
FIG. 31 shows a vector map of expression vector SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-3' UTR [2hBGpA-A120] -SS.
FIG. 32 shows a vector map of expression vector SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3' UTR [2hBGpA-A120] -SS.
FIG. 33 shows a vector map of expression vector SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3' UTR [2hBGpA-A120] -SS.
FIG. 34 shows a vector map of expression vector SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-huMAR-3' UTR [2hBGpA-A120] -SS.
FIG. 35 shows a vector map of the expression vector SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-huMAR-3' UTR [2hBGpA-A120] -SS.
FIG. 36 shows a vector map of expression vector SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3' UTR [2hBGpA-A120] -SS.
FIG. 37 shows a vector map of expression vector SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-huMAR-WPRE-3' UTR [2hBGpA-A120] -SS.
FIG. 38 shows a vector map of expression vector SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-huMAR-WPRE-3' UTR [2hBGpA-A120] -SS.
FIG. 39 shows a line graph of luciferase activity as indicated by RLU luminescence in HEK-293 cell culture medium following electroporation of cells with the expression vectors of FIG. 2 (conventional pDNA CMV-U), FIG. 30 (A: CMV-U1-3' UTR), FIG. 31 (B: E1-CMV-U1-3' UTR) and FIG. 32 (C: E1-CMV-U1-WPRE-3' UTR). * =p <0.05 and =p <0.01.
FIG. 40 shows a plot of relative luciferase intensities in HEK-293 cells of passages 1, 2, 3, 4 and 5 after electroporation of cells with the expression vector described in FIG. 39 on day 0 once every 7 days. * =p <0.05 and =p <0.01.
FIG. 41 shows a vector map of the expression vector pGL 2-CAG-SecNLuc-2A-eGFP-WPRE-bGlobal polyA.
FIG. 42 shows a vector map of expression vector 4-1pGL 2-SS-CAG [ CMV enhancer+CBA promoter+intron ] -secNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS.
FIG. 43 shows a vector map of expression vector 4-2pGL 2-SS-CAG [ E1X3+ CBA promoter+ intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS.
FIG. 44 shows a vector map of expression vector 4-3pGL 2-SS-CAG [ E2 (U100) +CBA promoter+intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS.
FIG. 45 shows a vector map of expression vector 4-4pGL 2-SS-CAG [ E1X3+ CBA promoter +UTR1] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS.
FIG. 46 shows a vector map of the expression vector 4-5-pGL 2-SS-CAG [ E2 (U100) +CBA promoter +UTR1] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS.
FIG. 47 shows a vector map of the expression vector 4-6-pGL 2-SS-CMV enhancer-EF 1-UTR1-SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS.
FIG. 48 shows bar graphs of luciferase activity indicated by RLU luminescence in HEK-293 cell medium 3 and 6 days after transfection with the expression vectors shown in FIG. 41 (conventional no SSeq pDNA CAG-W) and FIGS. 42-47 (4-1 to 4-6, respectively).
FIG. 49 shows an exemplary Sequence diagram of a self-limiting CRISPR gene editing system comprising flanking Super sequences (SSeq), synthetic enhancer (E1), CMV promoter (P CMV ) Optimized inclusion with tRNA-gRNA-PAM insertionSynthetic 5' UTR of the introns (UTR-tRNA-gRNA-PAM-1), casβ2 gene, and 3' UTR comprising human β -globin polyadenylation signal and gRNA-PAM insertion (HBg 3' UTR-gRNA-PAM).
Fig. 50 shows a schematic representation of self-limiting Cas expression from the sequence of fig. 52 during Homology Directed Repair (HDR) of chromosomal DNA with therapeutic GOI flanked by homology arms.
FIG. 51 shows a schematic diagram of two gene editing scenarios using a self-limiting CRISPR gene editing system. In scenario 1, msDNA containing a human expression cassette (e.g., therapeutic GOI) is first transfected for transient expression, and then the gene editing system of fig. 42 is transfected for HDR knock-in. In scenario 2, HDR knock-in is mediated by a single msDNA comprising a self-limiting CRISPR gene editing system and a human expression cassette flanked by homology arms.
Detailed Description
The present disclosure provides expression vectors, bacterial sequence-free vectors (e.g., ministring DNA (msDNA)), vector production systems, methods of making bacterial sequence-free vectors, compositions, and uses thereof.
All publications cited herein are incorporated by reference in their entirety, including but not limited to all journal articles, books, manuals, patent applications, and patents, cited herein to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.
I. Terminology
For easier understanding of the present disclosure, certain terms are first defined. As used in this application, each of the following terms shall have the following meanings, unless the context clearly indicates otherwise. Other definitions are set forth throughout the application.
It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.
The term "and/or" as used herein is to be taken to specifically disclose each specified feature or component, or a combination thereof with other specified features or components. Thus, the term "and/or" as used in a phrase such as "a and/or B" is intended to include "a and B", "a or B", "a" (alone) and "B" (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).
It should be understood that in any event that the present aspects are described herein by the expression "comprising," similar aspects are provided herein by the expression "consisting of and/or" consisting essentially of.
The term "about" or "substantially comprising" means that, for a particular value or composition, the value or composition determined by one of ordinary skill in the art to be within an acceptable error range depends in part on the manner in which the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" may mean within 1 or more standard deviations in accordance with the practice of the art. Alternatively, "about" or "substantially comprising" may mean a range of no more than 10% (i.e., ±10%). Furthermore, these terms may denote values of no more than an order of magnitude or no more than a factor of 5, particularly for biological systems or methods. When a particular value or composition is provided in the application and claims, unless otherwise indicated, "about" or "substantially comprising" should be construed to mean within an acceptable error range for the particular value or composition.
Any concentration range, percentage range, ratio range, or integer range is understood herein to include the values of any integer within the range, as well as fractions thereof (e.g., tenths and hundredths of integers) as appropriate, unless otherwise indicated. The numerical range includes the numbers defining the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, juo, pei-Show,2nd ed.,2002, CRC Press; the Dictionary of Cell and Molecular Biology,5th ed.,2013,Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology,2006,Oxford University Press provide a general dictionary of many terms to be used in the present disclosure to the skilled artisan.
Units, prefixes, and symbols are expressed in terms of their international unit System (SI) acceptance.
Unless otherwise indicated, nucleotide sequences are written in the 5 'to 3' direction from left to right. The amino acid sequence is written left to right in the amino to carboxyl direction.
The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.
An "amino acid" is a molecule having the structure in which a central carbon atom (α -carbon atom) is attached to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as the "carboxyl carbon atom"), an amino group (the nitrogen atom of which is referred to herein as the "amino nitrogen atom"), and a side chain group R. When incorporated into a peptide, polypeptide or protein, an amino acid loses one or more atoms of its amino acid carboxyl group in a dehydration reaction that links one amino acid to another amino acid. Thus, when incorporated into a protein, an amino acid is referred to as an "amino acid residue".
"protein" or "polypeptide" refers to any polymer of two or more (natural or unnatural) amino acids joined by peptide bonds, where the peptide bond is formed when the carboxyl carbon atom of an amino acid (or amino acid residue) of an alpha-carbon bonded carboxylic acid group is covalently bonded to the amino nitrogen atom of the alpha-carbon bonded amino group of an adjacent amino acid. The terms "protein" and "polypeptide" are used interchangeably herein. Similarly, fragments of proteins and polypeptides are also within the scope of the present disclosure and may be referred to herein as "proteins" or "polypeptides. In one aspect of the disclosure, the polypeptide comprises a chimera of two or more parent peptide fragments or proteins. The term "polypeptide" also means and encompasses post-translational modification ("PTM") products of polypeptides, including, but not limited to, disulfide bond formation, glycosylation, carbamylation, lipidation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, modification by non-naturally occurring amino acids, or any other manipulation or modification, e.g., conjugation to a labeling component. The polypeptides may be derived from natural biological sources or produced by recombinant techniques without having to be translated from the specified nucleic acid sequences. The polypeptide may be produced in any manner, including by chemical synthesis. An "isolated" polypeptide or fragment, variant or derivative thereof refers to a polypeptide that is not in its natural environment. There is no requirement for a specific level of purification. For example, an isolated polypeptide may simply be removed from its natural or natural environment. For the purposes of this disclosure, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as well as native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique.
A recombinant polypeptide comprising two or more proteins of the disclosure (i.e., a recombinant protein) may be encoded by a single coding sequence, wherein the single coding sequence comprises a polynucleotide sequence encoding each protein. Unless otherwise indicated, polynucleotide sequences encoding each protein are "in-frame" such that a single mRNA comprising these polynucleotide sequences will, upon translation, produce a single polypeptide comprising each protein. Typically, the proteins in a recombinant polypeptide as described herein may be fused to each other directly or may be separated by a peptide linker. Various polynucleotide sequences encoding peptide linkers are known in the art and include, for example, self-cleaving peptides.
Herein, "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides. In some cases, the polynucleotide comprises a sequence that is not directly contiguous with the coding sequence, or is directly contiguous (at the 5 'end or at the 3' end) with the coding sequence in the native genome of the organism from which it is derived. Thus, the term includes, for example, recombinant DNA incorporated into a vector, into an autonomously replicating plasmid or virus, into the genomic DNA of a prokaryote or eukaryote, or recombinant DNA which exists as a separate molecule (e.g., cDNA) independent of other sequences. The nucleotides of the present disclosure may be ribonucleotides, deoxyribonucleotides or modified forms thereof. Polynucleotides as used herein refer in particular to single-and double-stranded DNA, DNA in which single-and double-stranded regions are mixed, single-and double-stranded RNA, and RNA in which single-and double-stranded regions are mixed, hybrid molecules comprising DNA and RNA (which may be single-stranded or more typically double-stranded, or a mixture of single-and double-stranded regions). The term polynucleotide encompasses genomic DNA or RNA (depending on the organism, i.e., the RNA genome of the virus), as well as mRNA and cDNA encoded by genomic DNA. In certain aspects, the polynucleotide comprises a conventional phosphodiester linkage or an unconventional linkage (e.g., an amide linkage, as in Peptide Nucleic Acid (PNA)). An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, such as DNA or RNA, that has been removed from its natural environment. For example, for the purposes of this disclosure, a nucleic acid molecule comprising a polynucleotide encoding a recombinant polypeptide in a vector is considered "isolated". Other examples of isolated polynucleotides include: recombinant polynucleotides maintained in heterologous host cells or (partially or substantially) purified from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure also include synthetically produced polynucleotides and nucleic acids (e.g., nucleic acid molecules).
As used herein, "coding region" or "coding sequence" refers to a portion of a polynucleotide that consists of codons that can be translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not normally translated into an amino acid, it can be considered as part of the coding region; however, any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of the coding region are generally determined by a start codon at the 5 'end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes the carboxy terminus of the resulting polypeptide.
As used herein, an "expression cassette" comprises a nucleic acid sequence of interest (e.g., a nucleic acid sequence for expressing a polypeptide, DNA, or RNA) and an expression control region.
As used herein, "transgene" may be used interchangeably with "gene of interest (GOI)" and refers to the portion of a polynucleotide containing codons that can be translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not normally translated into an amino acid, it can be considered as part of a transgene; however, any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the transgene. The boundaries of the transgene are typically determined by a start codon at the 5 'end, which encodes the amino terminus of the resulting polypeptide, and a translation stop codon at the 3' end, which encodes the carboxy terminus of the resulting polypeptide.
As used herein, the term "expression control region" refers to a transcriptional control element operably linked to a coding region to direct or control the expression of a product encoded by the coding region, including, for example, cis-regulatory elements (CRM), promoters (e.g., tissue specific promoters and/or inducible promoters), enhancers, operators, repressors, ribosome binding sites, translational leader sequences, introns, post-transcriptional elements, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, and transcriptional termination signals, miRNA binding sites, and combinations thereof. Expression control regions include nucleotide sequences located upstream (5 '), internal or downstream (3') of the nucleic acid sequence of interest that can affect transcription, RNA processing, stability or translation of the relevant nucleic acid sequence of interest. If it is intended to express a transgene in eukaryotic cells, polyadenylation signals and transcription termination sequences are typically located 3' of the transgene.
When the coding region and the promoter are "operably linked" (i.e., are "operably linked"), induction of the function of the promoter will result in transcription of the mRNA comprising the coding region encoding the product, and the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct expression of the encoded product by the coding region nor with the ability of the DNA template to be transcribed. Expression control regions include nucleotide sequences located upstream (5 'non-coding sequences), internal or downstream (3' non-coding sequences) of the coding region, and which can affect transcription, RNA processing, stability or translation of the relevant coding region. If the coding region is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence will typically be located 3' of the coding sequence.
As used herein, the terms "host cell" and "cell" are used interchangeably and may refer to any type of cell or population of cells containing or capable of containing a nucleic acid molecule (e.g., a recombinant nucleic acid molecule), such as a primary cell, a cultured cell, or a cell from a cell line. The host cell may be a prokaryotic cell, or alternatively, the host cell may be a eukaryotic cell, such as a fungal cell, such as a yeast cell, and various animal cells, such as an insect cell or a mammalian cell.
As used herein, "culturing" and "cultured" refer to incubating cells under in vitro conditions that allow the cells to grow or divide or maintain the cells in a viable state. As used herein, "cultured cells" refers to cells that proliferate in vitro.
"individual" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as mammals, birds, pets, farm animals, non-human primates, sheep, cattle, goats, pigs, chickens, dogs, cats, and rodents such as mice, rats, and guinea pigs. In a preferred aspect, the individual is a human. The terms "individual" and "patient" are used interchangeably herein.
"administering" refers to physically introducing a therapeutic agent into an individual using any of a variety of methods and delivery systems known to those of skill in the art.
The terms "treat," "therapeutic," or "treatment" as used herein refer to any type of intervention or manipulation performed on an individual, or administration of an active agent to an individual, to reverse, reduce, ameliorate, inhibit or slow or prevent the progression, development, severity or recurrence of symptoms, complications, disorders, or biochemical indicators associated with a disease, or to increase overall survival. Treatment may be for individuals with a disease or individuals not with a disease (e.g., for prophylaxis, such as vaccination).
The terms "effective dose", "effective dose" or "effective amount" are defined as the amount of active agent sufficient to achieve, or at least partially achieve, the desired effect. By "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is meant any amount of drug that, when used alone or in combination with other therapeutic agents, promotes regression of the disease, which may be reflected in a decrease in severity of symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, an increase in overall survival (the length of time that a patient diagnosed with the disease remains alive from the date of diagnosis or the date of starting treatment of the disease), or prevention of injury or disability due to the disease. A therapeutically effective amount or dose of a drug includes a "prophylactically effective amount" or "prophylactically effective dose" that is any amount of drug that inhibits the occurrence or recurrence of a disease when the drug is administered alone or in combination with other therapeutic agents to an individual at risk of developing or recurrence of the disease. The ability of a therapeutic agent to promote regression of a disease or inhibit occurrence or recurrence of a disease can be assessed using a variety of methods known to those of skill in the art, such as in a human subject during a clinical trial, in an animal model system that predicts efficacy for human use, or by assaying the activity of an active agent in an in vitro assay.
Various aspects of the disclosure are described in further detail in the following subsections.
Expression vectors and vector production systems for producing vectors free of bacterial sequences
Bacterial-free sequence vectors and their production are described in U.S. patent nos. 9,290,778 and 9,862,954; nafissi and Slavcev, microbial Cell Factories 11:154 (2012); and Nafissi et al, nucleic Acids 3 (6): e165 (2014), the entire contents of which are incorporated herein by reference. These bacterial sequence-free vectors are produced from expression vectors (e.g., plasmids) comprising specific "supersequence" ("SS" or "SSeq") sites on both sides (i.e., on the 5 'and 3' sides) of an expression cassette comprising the target sequence for the recombinase, the expression cassette comprising the nucleic acid sequence(s) of interest. Specifically, each SS comprises a target sequence for a first recombinase and has additional target sequences for one or more additional recombinases integrated in the non-binding region of the first recombinase. When the expression vector is present in a recombinant cell expressing a suitable recombinase, the bacterial-free sequence vector containing the expression cassette will be isolated from the backbone DNA of the expression vector. To create a Circular Covalently Closed (CCC) bacterial sequence-free vector, the expression vector may be placed in a recombinant cell expressing a recombinase, such as Cre or Flp, which acts through its target sequence contained in SS. To generate a Linear Covalently Closed (LCC) bacterial sequence-free vector, also referred to herein as ministring DNA (msDNA), the expression vector may be placed in a recombinant cell expressing a recombinase, such as TelN or Tel, which acts through its target sequence contained in SS. The recombinantly produced bacterial sequence-free vector can then be purified from the cell and used directly as a delivery vehicle. See U.S. Pat. Nos. 9,290,778 and 9,862,954, nafissi and Slavcev, and Nafissi et al.
The msDNA vector with LCC-terminus is torsion-free and does not experience gyrase-directed negative supercoiled during production in e. Furthermore, integration of msDNA into the cell chromosome can result in chromosome breaks due to its double-stranded LCC topology, allowing the cell to be removed from the population. Thus, msDNA can eliminate any risk of insertional mutagenesis, protecting patients receiving msDNA from potential genotoxicity and cancer (Nafissi et al).
The present disclosure provides improved production of bacterial sequence-free vectors and improved bacterial sequence-free vectors. In some aspects, the production of a bacterial sequence-free vector is improved by removing contaminating expression vector sequences. In some aspects, the following aspects of the bacterial sequence-free vector are improved: improving the ability of the vector to build up in the cell (i.e., transfection efficiency), improving transgene expression (e.g., by combining enhanced transcription and translation), and improving expansion in the cell (e.g., vector replication and transmission to daughter cells).
In some aspects, the improvements of the present disclosure may be applicable to CCC or LCC vectors produced according to other methods known in the art.
A. Expression vector
Provided herein is an expression vector comprising: (a) a backbone sequence; (b) comprises the sequence: (i) an expression cassette comprising a nucleic acid sequence of interest, (ii) a first target sequence for a first recombinase located 5 'to the expression cassette, (iii) a second target sequence for the first recombinase located 3' to the expression cassette, and (iv) one or more additional target sequences for one or more additional recombinases, wherein the additional target sequences are integrated within the first and second target sequences, located in non-binding regions of the first recombinase: and (c) one or more of the following: (i) an endonuclease target sequence which is integrated within the first and/or second target sequences of the first recombinase and is located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the endonuclease target sequence is located between the backbone sequence and the cleavage site of the first recombinase and the one or more other recombinases, (ii) a synthetic enhancer comprising a nucleic acid sequence which is at least about 90% identical to SEQ ID NO:12, wherein the synthetic enhancer is integrated between the 3 'end of the first target sequence of the first recombinase and the 5' end of the other enhancer or promoter in the expression cassette, (iii) a Cytomegalovirus (CMV) enhancer integrated between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter in the expression cassette, (iv) a 5 'non-translated region (5' UTR) comprising an intron integrated between the promoter and the nucleic acid sequence of interest in the expression cassette, (iv) a polyadenylation signal sense element located between the polynucleotide and the polynucleotide of interest, and a polynucleotide sense gene (sense) a polynucleotide cassette located between the polynucleotide of interest, and a polynucleotide sense gene (sense gene) located between the polynucleotide of interest, or (viii) a DNA core targeting sequence (DTS) integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the DTS is located between the expression cassette and the cleavage sites of the first recombinase and the one or more other recombinases.
Reference herein to a "backbone" sequence is a sequence in an expression vector that is outside the expression cassette sequence and flanking SS sites, wherein the SS sites comprise the first and second target sequences of the first recombinase. The backbone sequence may include, for example, sequences for amplification and antibiotic selection of the expression vector in a host cell (e.g., E.coli), as described herein.
The "non-binding" region of a recombinase is a region in the target sequence of the first recombinase that is not acted upon (e.g., is not bound and/or cleaved by) the recombinase described herein.
A "cleavage site" of a recombinase is a site at which the recombinase initiates a double-strand break or single-strand nick in DNA associated with recombination.
In some aspects, the expression vector comprises an endonuclease target sequence located in the non-binding region of the first recombinase and the one or more other recombinases integrated within the first and/or second target sequences of the first recombinase, wherein the endonuclease target sequence is located between the backbone sequence and the cleavage site of the first recombinase and the one or more other recombinases. In some aspects, the endonuclease target sequence is integrated within the first target sequence of the first recombinase. In some aspects, the endonuclease target sequence is integrated within a second target sequence of the first recombinase. In some aspects, the endonuclease target sequence is integrated within the first and second target sequences of the first recombinase. In some aspects, the same endonuclease target sequence is integrated within the first and second target sequences of the first recombinase. In some aspects, the endonuclease target sequences integrated within the first and second target sequences of the first recombinase are for the same endonuclease. In some aspects, the endonuclease target sequence integrated within the first target sequence of the first recombinase is different from the endonuclease target sequence integrated within the second target sequence of the first recombinase. In some aspects, an endonuclease target sequence that is integrated within a first target sequence of a first recombinase and an endonuclease target sequence that is integrated within a second target sequence of the first recombinase are used for different endonucleases.
The presence of the endonuclease target sequence between the backbone sequence and the recombinase cleavage site in the expression vector ensures that the endonuclease target sequence remains linked to the backbone sequence after recombination as described, but not to the bacterial sequence-free vector. Thus, after recombination, the sequence comprising the backbone sequence and the endonuclease target site can be removed from the preparation comprising the bacterial sequence-free vector by exposure to the endonuclease, thereby reducing or avoiding the need for purification steps to remove the backbone sequence in the method of producing the bacterial sequence-free vector. In some aspects, the endonuclease is expressed post-recombination in a host cell of a vector production system as described herein, wherein the endonuclease cleaves DNA at an endonuclease target site, and the sequence comprising the backbone sequence and the endonuclease target site is degraded by an exonuclease (e.g., exonuclease V).
In some aspects, the expression vector comprises an endonuclease target sequence that homing endonucleases. In some aspects, the endonuclease target sequence is a sequence of I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-Dmo I, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SecIII, I-SceIV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-Tli, I-Tlip 061I or I-Vdi 141I. In some aspects, the endonuclease target sequence is for I-SceI. In some aspects, the endonuclease target sequence is for PI-SceI. The target sequence for homing endonucleases is well known in the art.
In some aspects, the expression vector comprises an endonuclease target sequence for an endonuclease used in genome editing (including endonucleases that are part of a nuclease genome editing system). In some aspects, the nuclease genome editing system is a clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, a transcription activator-like effector nuclease (TALEN) system, a Zinc Finger Nuclease (ZFN) system, or a meganuclease system.
In some aspects, the expression vector comprises an endonuclease target sequence for a Cas endonuclease. In some aspects, the Cas endonuclease is Cas9 (e.g., streptococcus pyogenes (Streptococcus pyogenes) Cas9 (SpCas 9), staphylococcus aureus (Staphlococcus aureus) Cas9 (SaCas 9), new francisbacterium (Francisella novicida) Cas9 (FnCas 9), or neisseria meningitidis (Neisseria meningitides) Cas9 (NmCas 9)), a Cas9 variant (e.g., cas9 beta 2, xcas9, spcas9-NG, spCas9-NRRH, spCas9-NRCH, spCas9-NRTH, spG, spRY), cas3, cas12 (e.g., cas12a, cas12b, cas12c, cas12d, or Cas12 e), cas13 (e.g., cas13a, cas13b, cas13c, or Cas13 d), or Cas14. In some aspects, the endonuclease target sequence of a Cas endonuclease as used herein is homologous to a guide RNA (gRNA) targeting sequence and includes a proto-spacer adjacent motif (PAM) recognized by the Cas endonuclease. The PAM site bearing sequences homologous to the gRNA targeting sequences can be routinely designed based on well known CRISPR systems. The gRNA may comprise a fusion of a targeting RNA (crRNA) sequence and a trans-activating RNA (tracrRNA) sequence that interact and function to guide the Cas endonuclease to the endonuclease target site and catalyze cleavage.
In some aspects, the expression vector comprises a synthetic enhancer comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 12 and is integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of another enhancer or promoter. In some aspects, the expression vector comprises a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO. 12 integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of another enhancer or promoter. In some aspects, the synthesis enhancer comprises multiple contiguous copies of the nucleic acid sequence, e.g., 1, 2, 3, 4, 5, or more contiguous copies. In some aspects, the synthesis enhancer comprises 3 consecutive copies of the nucleic acid sequence. In some aspects, the synthetic enhancer comprises a nucleotide sequence that hybridizes to SEQ ID NO:46 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the synthetic enhancer comprises SEQ ID NO: 46. In some aspects, the synthesis enhancer is integrated 5' to the chicken β -actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 47 is integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest. In some aspects, a chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 47 is integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a CMV enhancer integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleotide sequence that hybridizes to SEQ ID NO:12, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising the amino acid sequence of SEQ ID NO: 12. In some aspects, the CMV enhancer is integrated 3 'of multiple contiguous copies of the synthetic enhancer, e.g., at the 3' end of 1, 2, 3, 4, 5 or more contiguous copies of the synthetic enhancer. In certain aspects, the CMV enhancer is integrated 3' to 3 consecutive copies of the synthetic enhancer. In certain aspects, the CMV enhancer is integrated with SEQ ID NO:46 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In certain aspects, the CMV enhancer is integrated in SEQ ID NO:46, and a nucleic acid sequence 3' end thereof. In certain aspects, the CMV promoter is integrated 3 'of the CMV enhancer and 5' of the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a nucleotide sequence that hybridizes to SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO:38 or SEQ ID NO:39, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, which sequence is integrated between the first target sequence of the first recombinase and the nucleic acid sequence of interest. In some aspects, the expression vector comprises the sequence of SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO:38 or SEQ ID NO:39, and a nucleic acid sequence of seq id no. In some aspects, the polypeptide of SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO:38 or SEQ ID NO:39, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, or a nucleic acid sequence of SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. or SEQ ID NO:39, comprising all regulatory elements in the expression cassette 5' to the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a 5' utr comprising an intron, wherein the 5' utr (i.e., the 5' utr comprising the intron) is integrated in the expression cassette between the promoter and the nucleic acid sequence of interest.
In some aspects, the 5'utr improves splicing and translation of a transgene transcript from an expression vector or from a bacterial sequence-free vector produced by the expression vector as compared to the same expression vector or bacterial sequence-free vector lacking the 5' utr.
In some aspects, the intron comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the intron comprises SEQ ID NO:1, and a nucleic acid sequence of seq id no.
In some aspects, the 5' UTR comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 2, wherein SEQ ID NO. 2 is an optimized 5' UTR having an internal minimal intron, also referred to herein as "5' UTR1". In some aspects, the 5' utr comprises SEQ ID NO:2, and a nucleic acid sequence of seq id no.
In some aspects, the 5' UTR comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 4. In some aspects, the 5' utr comprises SEQ ID NO: 4.
In some aspects, the 5' utr further comprises a non-coding sequence integrated into the intron.
In some aspects, the intron has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% >, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID No. 1, or comprises SEQ ID No. 1, and the non-coding sequence is integrated between two nucleotides in the intron, the two nucleotides corresponding to SEQ ID NO:1 to any two nucleotides from position 25 to 55.
In some aspects, the non-coding sequence is non-prokaryotic and non-viral. In some aspects, the non-coding sequence is a eukaryotic sequence. In some aspects, the non-coding sequence comprises an intron, a ubiquitous chromatin opening element (ubiquitous chromatin opening element, UCOE), an S/MAR, an SV40 enhancer sequence (e.g., one or more SV40 enhancer sequences, such as two, three, four, five or more SV40 enhancer sequences), a vertebrate chromatin insulator (e.g., cHS 4), WPRE, or any combination thereof.
In some aspects, the non-coding sequence comprises an S/MAR. In some aspects, the S/MAR is MAR-5, provided herein as SEQ ID NO 9.
In some aspects, the 5' UTR comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 3. In some aspects, the 5' UTR comprises SEQ ID NO. 3.
In some aspects, the 5' UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 5. In some aspects, the 5' UTR comprises SEQ ID NO. 5.
In some aspects, the 5' utr is integrated in an expression cassette between a chicken β -actin promoter and a nucleic acid sequence of interest.
In some aspects, the 5' utr is integrated in the expression cassette between the CMV promoter and the nucleic acid sequence of interest.
In some aspects, the 5'utr is integrated in the expression cassette between the promoter and the nucleic acid sequence of interest, wherein the promoter is integrated 3' to the CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 12. In some aspects, the CMV enhancer is integrated 3' to the synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO. 12. In some aspects, the CMV enhancer is integrated 3 'of multiple contiguous copies of the synthetic enhancer, e.g., integrated 3' of 1, 2, 3, 4, 5 or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated 3' to 3 consecutive copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated with SEQ ID NO:46 has a 3' end of a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the CMV enhancer is integrated in SEQ ID NO:46, and a nucleic acid sequence 3' end thereof.
In some aspects, the expression vector comprises a polyadenylation signal integrated 3' to the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a Xenopus laevis (Xenopus leave) beta-globin polyadenylation signal, a human beta-globin polyadenylation signal, or a hybrid Xenopus laevis and human beta-globin polyadenylation signal. In some aspects, the polyadenylation signal comprises multiple copies of the xenopus laevis beta-globin polyadenylation signal, the human beta-globin polyadenylation signal, or a hybrid xenopus and human beta-globin polyadenylation signal, e.g., 1, 2, 3, 4, or 5 copies. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15. In some aspects, the polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO. 13, SEQ ID NO. 14 or SEQ ID NO. 15. In some aspects, the polyadenylation tail (i.e., poly (a) tail) is located 3' of the polyadenylation signal. In some aspects, the poly (a) tail is 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or more residues in length. In some aspects, a sequence comprising a polyadenylation signal and a poly (A) tail has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 18. In some aspects, the sequence comprising a polyadenylation signal and a poly (A) tail comprises SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18.
In some aspects, the expression vector comprises a vertebrate chromatin insulator in the expression cassette. In some aspects, the vertebrate chromatin insulator is a 5' -HS4 chicken- β -globin insulator (cHS 4). See, e.g., benambdellah et al, PLoS ONE 9 (1): e84268 (2014); lu et al, FEBS Open Bio 10:644-656 (2020); hanawa et al, mol. Ther.17 (4): 667-674 (2009); walters et al, mol. Cell. Biol.19 (5): 3714-3726 (1999). In some aspects, the vertebrate chromatin insulator is integrated in an expression cassette between a nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the vertebrate chromatin insulator is integrated in an intron of a 5' utr as described herein.
In some aspects, the vertebrate chromatin insulator comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID No. 8. In some aspects, the vertebrate chromatin insulator comprises SEQ ID NO. 8.
In some aspects, the vertebrate chromatin insulator improves the establishment of an expression vector or a bacterial-free sequence vector produced by the expression vector (i.e., transfection efficiency) as compared to the same expression vector or a bacterial-free sequence vector lacking the vertebrate chromatin insulator.
In some aspects, the expression vector comprises WPRE in the expression cassette. See, e.g., higashimoto et al, gene Therapy 14:1298-1304 (2007). In some aspects, the WPRE is integrated in an expression cassette between a nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the WPRE is integrated in an expression cassette at the 3 'end of the S/MAR described herein and at the 5' end of the polyadenylation signal described herein. In some aspects, the WPRE is integrated in an intron of a 5' utr as described herein.
In some aspects, WPRE comprises a nucleotide sequence that hybridizes to SEQ ID NO:11, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, WPRE comprises SEQ ID NO:11.
in some aspects, WPRE improves transgene expression from the expression vector or from a bacterial sequence-free vector generated from the expression vector as compared to the same expression vector or bacterial sequence-free vector lacking WPRE.
In some aspects, the expression vector comprises an S/MAR in the expression cassette. See, e.g., martens et al, mol.cell.biol.22 (8): 2598-2606 (2002); narwade et al, nucleic Acids Res.47 (14): 7247-7261 (2019). In some aspects, the S/MAR integration is located in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the S/MAR integration is located in the expression cassette at the 3 'end of the nucleic acid sequence of interest and at the 5' end of the WPRE described herein. In some aspects, the S/MAR is integrated in an intron of the 5' utr as described herein.
In some aspects, the S/MAR is MAR-3, MAR-4 or MAR-5, which are fragments of human interferon-beta MAR. See, e.g., wang et al, mol. Biol. Cell 30:2761-2770 (2019). In some aspects, the S/MAR comprises a sequence identical to SEQ ID NO:9, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the S/MAR comprises SEQ ID NO 9.
In some aspects, the S/MAR is a human cytotoxic serine protease-B (CSP-B) MAR or a CSP-C MAR. See, e.g., hanson and Ley, blood 79 (3): 610-618 (1992); klein et al, tissue anti-genes 35 (5): 220-228 (1990). In some aspects, the S/MAR comprises a sequence identical to SEQ ID NO:10 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the S/MAR comprises SEQ ID NO 10.
In some aspects, the S/MAR improves the expression level, stability, and/or persistence of the expression vector or the bacterial sequence-free vector (produced by the expression vector) as compared to the same expression vector or a bacterial sequence-free vector lacking the S/MAR (e.g., by episomal maintenance and replication, e.g., expansion of the vector and partitioning to daughter cells, and/or by preventing epigenetic silencing).
In some aspects, the expression vector comprising any one or more of (c) (i) - (c) (vii) as described above (i.e., without DTS) further comprises enhancer sequences flanking each side of the first and second target sequences of the first recombinase. In some aspects, the enhancer sequences flanking each of the first and second target sequences of the first recombinase are at least two enhancer sequences flanking each of the first and second target sequences of the first recombinase. In some aspects, the enhancer sequence is an SV40 enhancer sequence.
In some aspects, the expression vector comprises DTS. In some aspects, the DTS is integrated in the non-binding region of the first recombinase and the one or more other recombinases in the first and/or second target sequences of the first recombinase, wherein the DTS is located between the expression cassette and the cleavage site of the first recombinase and the one or more other recombinases. In some aspects, the DTS is an SV40 enhancer sequence. In some aspects, the DTS is cell specific. In some aspects, the DTS is specific for smooth muscle cells, embryonic stem cells, type II lung cells, endothelial cells, or osteoblasts.
The location of the DTS in the expression vector between the expression cassette and the cleavage site of the recombinase ensures that the DTS remains linked to the bacterial sequence-free vector, rather than to the backbone sequence, after recombination as described herein.
In some aspects, the expression vector comprises UCOE in the expression cassette. See, e.g., muller-Kuller et al, nucleic Acids Res.43 (3): 1577-1592 (2015); skip per et al, BMC Biotechnol.19:75 (2019); rudina et al, bioRxiv, doi.org/10.1101/626713 (2019); neville et al, biotechnol. Adv.35 (5): 557-564 (2017). In some aspects, the UCOE is located in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter or any enhancer. In some aspects, the UCOE is integrated in an intron of a 5' utr as described herein.
In certain aspects, the UCOE is an A2UCOE. In certain aspects, the UCOE comprises a nucleotide sequence that hybridizes to SEQ ID NO:6 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the UCOE is SEQ ID NO. 6.
In some aspects, the UCOE is an SRF-UCOE. See, for example, international publication No. WO2020223160. In some aspects, the UCOE comprises a nucleotide sequence that hybridizes to SEQ ID NO:7 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the UCOE is SEQ ID NO:7.
In some aspects, the UCOE improves transgene expression from the expression vector or from a bacterial sequence-free vector generated from the expression vector as compared to the same expression vector or a bacterial sequence-free vector lacking the UCOE.
In some aspects, the expression vector comprises an enhancer-1 in the expression cassette. In some aspects, enhancer-1 is integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter or any other enhancer. In some aspects, enhancer-1 is integrated between the 3 'end of the UCOE and the 5' end of the CMV enhancer. In some aspects, enhancer-1 comprises a nucleotide sequence that hybridizes to SEQ ID NO:12 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, enhancer-1 is SEQ ID NO:12.
in some aspects, the expression vector comprises a CMV, EFl, SV, CAG, rho, VDM2, HCR or HLP promoter, or variant thereof, in the expression cassette. In some aspects, the expression vector comprises a CMV promoter variant in the expression cassette. See, for example, international publication No. WO2012099540; xu et al, bioengineered 10 (1): 548-560, DOI:10.1080/21655979.2019.1684863 (2019).
In some aspects, the expression vector comprises the EFl- α promoter in an expression cassette. In some aspects, the expression vector comprises a CMV enhancer and an EF 1-a promoter in the expression cassette.
In some aspects, the expression vector comprises a 3'utr in the expression cassette, the 3' utr comprising two copies of the β -globin polyadenylation signal. In some aspects, the 3'utr is integrated between the nucleic acid sequence of interest and the 5' end of the second target sequence of the first recombinase.
In some aspects, the 3' utr comprises two copies of a xenopus laevis β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:13 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:13.
in some aspects, the 3' utr comprises two copies of a human β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:14, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:14.
In some aspects, the 3' utr comprises one copy of a xenopus laevis β -globin polyadenylation signal and one copy of a human β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:15, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:15.
in some aspects, the 3' utr further comprises a poly (a) tail (i.e., at the 3' end of the 3' utr) comprising 100 to 120 adenine nucleotides, i.e., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 adenine nucleotides.
In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:16 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:16.
in some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:17 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:17.
In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:18, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:18.
the expression vector may comprise any combination of the above modifications to the first and/or second target sequences and/or expression cassettes described herein. In some aspects, the combination provides a synergistic effect.
In some aspects, the first and second target sequences of the first recombinase and the one or more other target sequences of the one or more other recombinases are selected from the group consisting of: PY54 pal site, N15 telRL site, loxP site,telRL site, FRT site, phiC31 attP site and lambda attP site. In some aspects, the expression vector comprises all of these target sequences. In some aspects, the expression vector comprises a pal site and telRL, loxP, and FRT recombinase target binding sequences integrated in the pal site. In some aspects, the first and second target sequences of the first recombinase each comprise the nucleic acid sequence of SEQ ID NO. 33.
In some aspects, in any of the expression cassettes described herein, the nucleic acid sequence of interest may comprise a sequence encoding: polypeptides, RNAs (messenger RNAs (mrnas), micrornas (mirnas), small interfering RNAs (sirnas)), small hairpin RNAs (shrnas), ribozymes, or antisense RNAs), or non-coding DNA (e.g., antisense oligonucleotides). In some aspects, the nucleic acid sequence of interest is a genomic DNA sequence comprising introns and/or exons. In some aspects, the nucleic acid sequence of interest comprises a sequence encoding: an anti-cancer agent, a tumor suppressor, an apoptotic agent, an anti-angiogenic agent, an enzyme, a cytotoxic agent, a suicide gene, a cytokine, an interferon, an interleukin, an immunomodulator, an immunostimulant, an immunosuppressant, a chemokine, an antigen for stimulating antigen presenting cells, an antibody (e.g., a heavy and/or light chain of an antibody, such as a monoclonal, chimeric, humanized or human antibody, or antigen binding fragment thereof), a genome editing system or portion thereof (e.g., CRISPR-Cas, TALEN, ZFN, or a meganuclease system or portion thereof, such as Cas endonuclease or gRNA), or an immunogenic agent (e.g., as a VLP and/or vaccine). In some aspects, the nucleic acid sequence of interest comprises a sequence encoding a polypeptide that is capable of forming a VLP when the nucleic acid sequence is expressed in a cell.
Exemplary therapeutic targets and indications include: genes associated with monogenic disorders, including disorders such as liver, blood or eye, galactosidase alpha (GLA, e.g. for the treatment of Fabry disease), sodium-voltage-gated channel alpha subunit 1 (SCN 1A, e.g. for the treatment of Dravet syndrome), ATP-binding cassette a subfamily member 4 (ABCA 4, e.g. for the treatment of Stargardt disease), surface-active protein B (SP-B, e.g. for the treatment of surface-active substance dysfunction), surface-active protein C (SP-C, e.g. for the treatment of surface-active substance dysfunction), ATP-binding cassette a subfamily member 3 (ABCA 3, e.g. for the treatment of surface-active substance dysfunction), solute carrier family 34 member 2 (SLC 34A2, e.g. for the treatment of alveolar and/or testicular microcompus), cystic fibrosis transmembrane conductance regulator (CFTR), e.g. for the treatment of cystic fibrosis), glutamate decarboxylase (GAD, e.g. GAD65 or GAD67, e.g. for the treatment of parkinson disease), aspartate-gene (ASPA, also known as aminoacylase (ASPA), e.g. for the treatment of surface-active substance dysfunction), surface-active substance C (SP-C, e.g. for the treatment of 2, e.g. for the treatment of the deficiency of the neurite, the same factor (aap), the treatment of the growth factor (e.g. for the growth factor (TPP), the same factor (e.g. for the growth factor (tsp), the treatment of the neurite, tsn), the factor (aap) or the protein factor (e.g. for the growth factor (tsf), the protein factor (e.g. is derived from the factor (aatsi), also known as neuronal ceroid lipofuscinosis type 2 (CLN 2), e.g. for the treatment of Batten's disease, e.g. CLN 2's disease), arylsulfatase a (ARSA, e.g. for the treatment of metachromatic leukodystrophy), N-sulfoglucosamine sulfohydrolase (SGSH, e.g. for the treatment of Sanfilippo syndrome, type a), sulfatase modification factor 1 (SUMF 1, e.g. for the treatment of Sanfilippo syndrome, type a), N-acetyl-alpha-glucosaminidase (NAGLU, e.g. for the treatment of Sanfilippo syndrome, type B), motor neuron survival 1 (SMN 1, e.g. for the treatment of spinal muscular dystrophy 1), retinal pigment epithelium-specific 65kDa protein (RPE 65, also known as retinoid isomerase, e.g. for the treatment of Leber congenital melanosis), rab 1 (REP 1, e.g. for the treatment of anemic membrane), retinal split 1 (reosin, e.g. for the treatment of SGs), SG-alpha-glucosidian, SG-alpha-aminoglycans (e.g. for the treatment of dysmyo), levan (e.g. for the treatment of dysmyo-type β), levan (e.g. for the treatment of dysmyo- β), levan (e.g. for the treatment of dysmyo-type β), or for the treatment of dysmyometria (e.g. dysmyo), levan (e.g. for the dysmyo- β -type β, e.g. dysmyotone (e.g. dysmyometria) or (e.g. dysmyometria) and for the treatment of the dysone, for the treatment of familial LPL deficiency), acid alpha-glucosidase (GAA, e.g. for the treatment of pompe disease), tumor necrosis factor receptor Fc (TNFR: fc, e.g. for the treatment of arthritis, e.g. inflammatory arthritis), sarcoplasmic/endoplasmic reticulum Ca (2+) ATPase 2a (SERCA 2a, e.g. for the treatment of congestive heart failure), factor VIII or factor IX (FVIII or FIX, e.g. for the treatment of hemophilia B), porphobilinogen deaminase gene (PBGD, e.g. for the treatment of acute intermittent porphyria), soluble FMS-like tyrosine kinase-1 (sFlt 1, e.g. for the treatment of age-related macular degeneration or cancer, e.g. ovarian cancer), soluble chimeric Vascular Endothelial Growth Factor (VEGF) receptor (e.g. comprising VEGFR-1 and VEGF-R2 domains. For treating cancer, such as melanoma or colon cancer), soluble VEGFR3 (e.g., for treating cancer, such as endometrial cancer), soluble VEGF-C decoy receptor (svgfr 3-Fc, e.g., for treating cancer, such as melanoma, renal cell carcinoma, or prostate cancer), pigment epithelium derived growth factor (PEDF, e.g., for treating cancer, such as Lewis lung cancer), neutralizing mab to VEGFR2 (e.g., DC101, e.g., for treating cancer, such as melanoma or glioblastoma), endostatin (e.g., for treating cancer, such as bladder cancer or pancreatic cancer), angiostatin (angiostatin, e.g., for treating cancer, such as liver cancer), both endostatin and angiostatin (i.e., as a bicistronic sequence, e.g., for treating cancer, such as ovarian cancer or prostate cancer), endostatin mutant (i.e.g., P1254A-endostatin, e.g., for treating cancer, such as ovarian cancer), anti-angiogenic domain (3 TSR, e.g., for treating cancer, such as pancreatic factor, such as for treating glioblastoma), TSP-1 (e.g., for treating cancer), a factor for use in the treatment of the pro-channel (e.g., for treating glioblastoma), for example, such as a tumor, or a tumor, a factor (e.g., for use in the UPR-5, a tumor, or a factor-pathway such as a tumor); such as IRE1 alpha, XBP-1 or ATF6, e.g., for the treatment of cancer, such as breast cancer, vasostatin (e.g., for the treatment of cancer, such as lung cancer), herpes simplex virus type 1 thymidine kinase (HSV-TK), such as for the treatment of cancer, such as breast cancer, sc39TK (e.g., for the treatment of cancer, such as cervical cancer), diphtheria Toxin A (DTA), such as for the treatment of cancer, such as cervical cancer or myeloma), p53 upregulated apoptosis-regulating agents (PUMA, such as for the treatment of cancer, such as cervical cancer or myeloma), tumor Necrosis Factor (TNF) -related apoptosis-inducing ligand (TRAIL, for the treatment of cancer, such as lymphoma, hepatocellular carcinoma, head and neck squamous cell carcinoma (i.e.g., head and neck cancer) or glioblastoma), soluble TRAIL (e.g., for the treatment of cancer, such as liver cancer or lung adenocarcinoma), IFN-beta (e.g., for the treatment of cancer, such as colorectal cancer, such as for example, colorectal cancer, lung cancer, such as, lung glioblastoma or glioblastoma multiforme), IFN-alpha (e.g., for the treatment of cancer, such as metastatic melanoma), CD40 (CD 40) or CD40 (IL-40) or IL-24, such as for the treatment of apoptosis gene-related IL (e.g., such as IL-24, e.g., for the treatment of apoptosis, such as IL-24 (e.g., IL-24), such as Mixed Lineage Leukemia (MLL)/AF 4 positive Acute Lymphoblastic Leukemia (ALL)), IL15 (e.g., for the treatment of cancer, such as metastatic hepatocellular carcinoma), secondary lymphoblastic chemokine (SLC, such as for the treatment of cancer, such as liver cancer), NK4 (N-terminal hairpin of Hepatocyte Growth Factor (HGF) and the subsequent four Kringle domains, such as for the treatment of cancer, such as metastatic Lewis lung cancer), tumor necrosis factor superfamily member 14 (TNFSF 14, also known as LIGHT, such as for the treatment of cancer, such as cervical cancer), granulocyte-macrophage colony stimulating factor (GM-CSF, such as for the treatment of cancer), TNF- α (e.g., for the treatment of cancer, such as glioma), dominant negative mutant of Survivin (e.g., C84A or T34A, such as for the treatment of cancer, such as colon cancer or gastric cancer), C-terminal fragment of human telomerase reverse transcriptase (hTC 27, such as for the treatment of cancer, such as glioblastoma), maspin (e.g., for the treatment of cancer, such as prostate cancer), nm23H1 (e.g., for the treatment of metastatic cancer, such as for the treatment of liver cancer, such as for the human colorectal cancer, such as for the treatment of human factor (FK 1, such as for the treatment of human factor E, such as human factor B1, such as for the human factor B1, for the anti-factor B1, for the treatment of human factor (FK 1, such as for the anti-factor 1, for the binding to human factor (FK 1, for the anti-F, for the anti-factor 1, for the anti-factor for the anti-tumor protein, for the anti-tumor protein for the treatment of human factor for the human factor for example for the human factor), the C-X-C motif chemokine receptor 2 (CXCR 2) C-tail sequence (e.g., for use in the treatment of cancer, such as pancreatic cancer), alpha-tocopherol related protein (TAP, e.g., for use in the treatment of cancer, such as prostate cancer), trichosanthin (Trichosanthin, e.g., for use in the treatment of cancer, such as hepatocellular carcinoma), decorin (decorin, e.g., for use in the treatment of cancer, such as glioblastoma multiforme), cathelicidin (e.g., for use in the treatment of cancer, such as colon cancer), niemann-Pcik type C2 (NPC 2, e.g., for use in the treatment of cancer, such as hepatocellular carcinoma), mullerian inhibitory substances (MISS, e.g., for use in the treatment of cancer, such as ovarian cancer), P53 (e.g., for use in the treatment of cancer, such as bronchioloalveolar carcinoma), shRNA against cancer highly expressed protein 1 (Hec 1, e.g. for the treatment of cancer, e.g. glioma), shRNA against Epstein-Barr virus latent membrane protein-1 (EBV LMP-1, e.g. for the treatment of cancer, e.g. nasopharyngeal carcinoma), antisense RNA against human papilloma virus 16E7 oncogene (HPV 16-E7, e.g. for the treatment of cancer, e.g. cervical cancer), shRNA against androgen receptor (AR, e.g. for the treatment of cancer, e.g. prostate cancer), siRNA against Snail (also called SNA1, e.g. for the treatment of cancer, e.g. pancreatic cancer), siRNA against Slug (i.e. protein product of SNAI2, e.g. for the treatment of cancer, e.g. cholangiocarcinoma (liver cancer)), shRNA against four half LIM-Only proteins 2 (FHL 2, for example for the treatment of cancer, such as colon cancer), miR-26a (for example for the treatment of cancer, such as hepatocellular carcinoma), HPV16 structural protein L1 (HPV 16-L1, for example for the treatment of cancer, such as cervical cancer), HPV16E5, E6 and E7 oncogenes (HPV 16E 5/E6/E7, for example for the treatment of cancer, such as cervical cancer), B-cell leukemia/lymphoma 1 (BLC 1) idiotypes (for example for the treatment of cancer, such as B-cell leukemia/lymphoma 1), EBV LMP1 and LMP2 fused to a heat shock protein (EBV LMP2/1-HSP, for example for the treatment of cancer, such as nasopharyngeal carcinoma), carcinoembryonic antigen (CEA, for example for the treatment of cancer, such as colon cancer), B/T lymphopoietin-attenuated soluble forms in combination with heat shock proteins (BTLA and HSP70, for example, for the treatment of cancer, such as melanoma lung metastasis), HPV16-L1/E7 (e.g., for the treatment of cancer, such as cervical cancer), HPV16-L1 (e.g., for the treatment of cancer, such as cervical cancer), anti-EGFR antibodies (e.g., 14D1, e.g., for the treatment of cancer, such as vulvar cancer), anti-death receptor 5 (DR 5) antibodies (e.g., adximab), for the treatment of cancer, such as liver cancer or colon cancer, anti-enolase 1 (ENOI 1) antibodies (e.g., for the treatment of cancer, such as pancreatic ductal adenocarcinoma), anti-VEGFA antibodies (e.g., bevacizumab, e.g., for the treatment of cancer, such as metastatic lung cancer or ovarian cancer), anti-cancer, such as metastatic lung cancer, ovarian cancer, and the like, mucin 1 (MUC 1) antigen (e.g., for the treatment of cancer (e.g., gastric cancer) or aquaporins (e.g., hAQP1, e.g., for the treatment of radiation-induced parotid salivary gland hypofunction, i.e., xerostomia).
In some aspects, the nucleic acid sequences of interest are used for gene editing (e.g., gene therapy, including treatment of genetic defects, disorders, or diseases).
In some aspects, the nucleic acid sequence of interest is used to insert a target site for gene editing (i.e., a site within a DNA or RNA sequence that is the target of gene editing). The target site for gene editing may comprise any genetic element, such as any cis-element. In some aspects, the target site for gene editing is located in an exon of a gene, an intron of a gene, or a regulatory element of a gene.
In some aspects, the gene editing comprises an endonuclease. In some aspects, the endonuclease is associated with a genome editing system. In some aspects, the endonuclease is, for example, a homing endonuclease, a site-specific nuclease, a structure-directed nuclease, or an RNA-directed nuclease (e.g., a transposon-encoded RNA-directed nuclease).
In some aspects, the gene editing comprises a genome editing system that creates double strand breaks within a target site of the gene editing. In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN or meganuclease gene editing system.
In some aspects, the nucleic acid sequence of interest is inserted into the target site of gene editing by non-homologous end joining at a double strand break. In some aspects, the double strand break is generated by a CRISPR-Cas system. In some aspects, the expression vectors described herein comprise a Cas endonuclease target sequence (i.e., a sequence homologous to a gRNA targeting sequence) located between the first and second target sequences of the first recombinase and the nucleic acid sequence of interest (i.e., between the 5 'super sequence and the nucleic acid sequence of interest and between the 3' super sequence and the nucleic acid sequence of interest), wherein the target site of gene editing (e.g., a target site in a chromosome) comprises the same Cas endonuclease target sequence. For example, processing a Cas endonuclease target sequence flanking a nucleic acid sequence in a bacterial-free sequence vector (e.g., msDNA) generated from an expression vector will result in removal of the super sequence, thereby rendering the linear covalently closed bacterial-free sequence vector, e.g., msDNA, linear and open ended, with reactive ends suitable for non-homologous end joining events.
In some aspects, the nucleic acid sequence of interest is inserted into the target site of gene editing by homology-directed repair, wherein the insertion is achieved by recombination between sequences flanking the double-strand break site and the homologous sequence to which the nucleic acid sequence of interest is linked.
In some aspects, the nucleic acid sequence of interest has sufficient homology to sequences flanking the double-strand break site to support homology directed repair.
In some aspects, the nucleic acid sequence of interest is flanked by 5 'and 3' homology arms (i.e., sequences that have sufficient homology to sequences flanking the double-strand break site to mediate homology-directed repair).
In some aspects, homology sufficient to mediate homology-directed repair includes at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% homology between the nucleic acid sequence of interest and sequences flanking the double-strand break site or between the homology arm of the nucleic acid sequence of interest and sequences flanking the double-strand break site. In some aspects, the sequence flanking the double-strand break site is within about 100 bases, about 90 bases, about 80 bases, about 70 bases, about 60 bases, about 50 bases, about 45 bases, about 40 bases, about 35 bases, about 30 bases, about 25 bases, about 20 bases, about 15 bases, about 10 bases, or about 5 bases of the double-strand break site, or immediately flanking the double-strand break site.
In some aspects, homology-directed repair is performed by a CRISPR-Cas system. In some aspects, the expression vectors described herein comprise a CRISPR-Cas system. In some aspects, the expression vector comprises a tRNA-gRNA polycistronic flanking each side of the sequence encoding the Cas endonuclease (e.g., immunosilent Cas9- β2). An exemplary aspect is shown in fig. 49. In some aspects, the expression vector comprises a 5'utr (e.g., 5' utr 1) as described herein, comprising a tRNA-gRNA polycistron in an intron. In some aspects, the expression vector comprises a chimeric intron described herein comprising a tRNA-gRNA polycistronic. In some aspects, the EF 1-alpha promoter described herein comprises a tRNA-gRNA polycistronic in an inherent intron. In some aspects, the polyadenylation signals or 3' UTRs described herein comprise tRNA-gRNA polycistronic. For example, when the Cas endonuclease is expressed from a vector comprising a flanking tRNA-gRNA polycistronic (i.e., from an expression vector or a bacterial sequence-free vector (e.g., msDNA)), the gRNA will be excised as free RNA and direct the Cas endonuclease to target sites of gene editing (e.g., target sites in the chromosome) as well as the flanking gRNA sites on the vector. This will result in self-restriction of the Cas endonuclease, limiting further expression of the Cas endonuclease from the vector. A schematic representation of this approach is shown in FIG. 50, which also shows homology directed repair mediated by a nucleic acid of interest (i.e., a gene of interest, GOI) flanked by homology arms on separate vectors. In some aspects, an expression vector as described herein comprises a nucleic acid sequence of interest flanked by homology arms, e.g., as shown in scene 1 of fig. 51. In some aspects, the nucleic acid sequence of interest and the self-limiting CRISPR-Cas system as described herein are located on a single expression vector as described herein, as shown in scenario 2 of fig. 51. In the latter aspect, the sequence comprising the self-limiting CRISPR-Cas system may be located 5' to the sequence comprising the nucleic acid sequence of interest flanked by homology arms.
In some aspects, the nucleic acid sequence of interest is homologous to a target site of gene editing and comprises one or more nucleotide insertions, deletions, inversions, or rearrangements compared to the target site. In some aspects, the nucleic acid of interest is a genomic sequence, coding region, exon, intron, or any portion thereof for replacing a homologous sequence at a target site.
In some aspects, the nucleic acid sequence of interest is non-homologous to a target site for gene editing.
In some aspects, the nucleic acid sequence of interest restores the function of a deletion, corrects an abnormality, or provides other functions associated with the target site of gene editing.
In some aspects, the nucleic acid sequence of interest is used to knock out gene expression (i.e., gene silencing) associated with a target site of gene editing.
In some aspects, the nucleic acid sequence of interest is used for in vivo gene editing.
In some aspects, the nucleic acid sequence of interest is used for in vitro gene editing.
In some aspects, the nucleic acid sequences of interest are used for ex vivo gene editing (e.g., cell therapies, such as Chimeric Antigen Receptor (CAR) T cell therapies).
In some aspects, the gene editing comprises epigenetic modification, and the expression vectors described herein comprise an epigenetic effector molecule as a nucleic acid of interest. In some aspects, the epigenetic effector molecule mediates, for example, acetylation or deacetylation, methylation or demethylation, or phosphorylation or dephosphorylation. In some aspects, the epigenetic effector molecule inhibits acetylation or deacetylation, methylation or demethylation, or phosphorylation or dephosphorylation. In some aspects, the epigenetic modification is a histone modification. In some aspects, the histone modification is histone acetylation and the nucleic acid of interest is histone acetyltransferase. In some aspects, the histone modification is histone deacetylation and the nucleic acid of interest is a histone deacetylase. In some aspects, the epigenetic modification is a DNA modification. In some aspects, the DNA modification is DNA methylation and the nucleic acid of interest is a DNA methylase. In some aspects, the DNA modification is DNA demethylation and the nucleic acid of interest is a DNA demethylase. In some aspects, the epigenetic effector molecule is fused to a targeting molecule, such as a DNA binding molecule, to target the effector molecule to a location on a chromosome.
In some aspects, the expression cassette is polygenic, i.e., the expression cassette comprises two or more nucleic acid sequences of interest encoding two or more polypeptides, respectively.
In some aspects, the expression cassette comprises a single open reading frame comprising a nucleic acid sequence encoding a self-cleaving peptide located between each nucleic acid sequence encoding a polypeptide, thereby allowing the translation product of the expression cassette to be cleaved into two or more polypeptides within the cell. In some aspects, the self-cleaving peptide is a 2A self-cleaving peptide. In some aspects, the 2A self-cleaving peptide is P2A from porcine teschovirus-1 (Porcine teschovirus-1). In some aspects, the 2A self-cleaving peptide is T2A from the vein occlusion virus (Thosea asigna virus) 2A. In some aspects, the self-cleaving peptide comprises any one or more of 2A, P a and T2A. In some aspects, the self-cleaving peptide comprises P2A and T2A.
In some aspects, the expression cassette further comprises a nucleic acid sequence encoding a gene expression marker. In some aspects, the marker of gene expression is a fluorescent reporter gene, e.gSuch as green fluorescent protein (GFP, e.g., enhanced GFP (eGFP)), red Fluorescent Protein (RFP), yellow Fluorescent Protein (YFP), or near infrared fluorescent protein (iRFP); bioluminescent reporter genes, such as luciferases (e.g., nanoluciferases, i.e. (NLuc), england et al, bioconjug.chem.27 (5): 1175-1187 (2016), promega Corporation); a selectable antibiotic marker; or LacZ. In some aspects, the expression cassette comprises a nucleic acid sequence encoding a self-cleaving peptide between a nucleic acid sequence encoding a gene expression marker and any other nucleic acid sequence encoding a polypeptide.
The expression cassette may contain any expression control region known to those of skill in the art operably linked to the nucleic acid sequence(s) of interest. In some aspects, the expression control region is a promoter, enhancer, operator, repressor gene, ribosome binding site, translational leader, intron, polyadenylation recognition sequence, RNA processing site, effector binding site, stem-loop structure, transcriptional termination signal, or a combination thereof.
In some aspects, the expression vector is used to produce a bacterial sequence-free vector. In some aspects, the bacterial sequence-free vector is a circular covalently closed vector. In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
B. Carrier production system
Provided herein are vector production systems comprising a recombinant cell encoding a recombinase under the control of an inducible promoter, wherein the recombinant cell comprises an expression vector as described herein, wherein the expression vector comprises first and second target sequences of a first recombinase and one or more other target sequences of one or more other recombinases, and wherein the recombinase targets one of the first and second target sequences of the first recombinase or the one or more other target sequences of the one or more other recombinases.
Suitable host cells for use in the vector production system include microbial cells, for example bacterial cells such as E.coli cells, and yeast cells such as Saccharomyces cerevisiae. Mammalian host cells, including Chinese Hamster Ovary (CHO) cells (e.g., of the K1 lineage (ATCC CCL 61) or Pro5 variant (ATCC CRL 1281)), may also be used; fibroblast-like cell lineages derived from SV40 transformed African green monkey kidney, CV-1 lineages (ATCC CCL 70), COS-1 lineages (ATCC CRL 1650) or COS-7 lineages (ATCC CRL 1651); murine L cells; murine 3T3 cells (ATCC CRL 1658); murine C127 cells; human embryonic kidney cells of the 293 lineage (ATCC CRL 1573); human cancer cells, including cancer cells of the HeLa lineage (ATCC CCL 2); the neuroblastoma cell line IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10) or SK-N-SH (ATCC HTB 11).
Suitable recombinases catalyze DNA exchange at the recombinase target sequences as described herein, including, but not limited to TelN, tel, tel (gp 26K 02 phage), cre, flp, phiC, int, and other lambdoid phage integrases, such as phi 80, HK022, and HP1 recombinases. In some aspects, the recombinase is TelN, tel, cre or Flp.
In some aspects, the recombinant cell further encodes an endonuclease under the control of an inducible promoter, wherein the endonuclease targets an endonuclease target sequence in an expression vector.
A suitable endonuclease cleaves the polynucleotide at the endonuclease target sequence. In some aspects, the endonuclease is a homing endonuclease. In some aspects, the homing endonuclease is I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-Dmo I, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SecIII, I-SceIV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-Tli, I-Tlip 141I, I-Tsp061I, or I-VdiI. In some aspects, the endonuclease is I-SceI. In some aspects, the endonuclease is PI-SceI. In some aspects, the recombinant cell encodes a nuclease genome editing system comprising an endonuclease. In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN or meganuclease system. In some aspects, the nuclease genome editing system is a class 1 or class 2 CRISPR-Cas system. In some aspects, the nuclease genome editing system is a type I, type II, type III, type IV, type V, or type VI CRISPR-Cas system. In some aspects, the Cas endonuclease in the CRISPR-Cas system is Cas9 (e.g., spCas9, saCas9, fnCas9, or NmCas 9), cas9 variant (e.g., cas beta 9, xcas9, spcas9-NG, spCas9-NRRH, spCas9-NRCH, spCas9-NRTH, spG, spRY), cas3, cas12 (e.g., cas12a, cas12b, cas12c, cas12d, or Cas12 e), cas13 (e.g., cas13a, cas13b, cas13c, or Cas13 d), or Cas14.
Recombinant host cells encoding a recombinase or a recombinase and an endonuclease can be prepared using well known techniques. For example, a nucleic acid sequence encoding a selected recombinase or endonuclease is introduced into a cell under suitable cell transformation conditions using a suitable vector. The recombinant host cell may be transformed by an expression vector or by integrating nucleic acid sequences encoding a recombinase and/or an endonuclease into the host cell genome. In aspects where the endonuclease is associated with a nuclease genome editing system, the host cell may be designed to encode all components of the nuclease genome editing system, for example, by transforming the host cell with one or more expression vectors comprising all components, or by integrating all components into the host cell genome, or by a combination of transformation and integration of components. In some aspects, the host cell encodes a Cas or Cas-like endonuclease and a gRNA.
Expression of a recombinase or endonuclease (including an endonuclease of a nuclease genome editing system) may be under the control of an inducible promoter (i.e., a promoter that is activated under specific physical or chemical conditions or stimuli). In some aspects, the inducible promoter is heat regulated, chemically regulated, IPTG regulated, glucose regulated, arabinose-inducible, T7 polymerase-regulated, cold shock-inducible, pH-inducible, or a combination thereof.
Provided herein are recombinant cells comprising an expression vector as described herein, the expression vector comprising first and second target sequences for a first recombinase and one or more other target sequences for one or more other recombinases. In some aspects, the recombinant cell encodes the first recombinase and/or one or more of the one or more recombinases described herein. In some aspects, the recombinant cells encode one or more endonucleases described herein. In some aspects, the recombinant cells encode a nuclease genome editing system described herein.
Provided herein is a method of producing a bacterial sequence-free vector comprising: the vector production system described herein is incubated under conditions suitable for expression of the recombinase. In some aspects, the method further comprises incubating the vector production system under conditions suitable for expression of the endonuclease encoded by the recombinant cell. In some aspects, the method further comprises incubating the vector production system under conditions suitable for expression of the nuclease genome editing system encoded by the recombinant cell. In some aspects, the method further comprises harvesting the bacterial sequence-free vector.
Provided herein are bacterial sequence-free vectors produced by the methods of producing bacterial sequence-free vectors described herein.
Bacterial sequence-free vector
Provided herein are bacterial sequence-free vectors comprising: (a) An expression cassette comprising a nucleic acid sequence of interest, and (b) one or more of: (i) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 12 located 5' to another enhancer or promoter in the expression cassette, (ii) a CMV enhancer located 5' to the promoter in the expression cassette, (iii) a 5' utr comprising an intron, wherein the 5' utr is integrated between the promoter and the nucleic acid sequence of interest in the expression cassette, (iv) a vertebrate chromatin insulator integrated between the nucleic acid of interest and the polyadenylation signal in the expression cassette, (v) a WPRE integrated between the nucleic acid of interest and the polyadenylation signal in the expression cassette, (vi) an S/MAR integrated between the nucleic acid of interest and the polyadenylation signal in the expression cassette, or (vii) a DTS located 5' to the expression cassette.
In some aspects, a bacterial-free sequence vector comprises a synthetic enhancer 5' to another enhancer or promoter in the expression cassette, the synthetic enhancer comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID No. 12. In some aspects, the bacterial sequence-free vector comprises a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO. 12 5' to another enhancer or promoter in the expression cassette. In some aspects, the synthesis enhancer comprises multiple contiguous copies of the nucleic acid sequence, e.g., 1, 2, 3, 4, 5, or more contiguous copies. In some aspects, the synthesis enhancer comprises 3 consecutive copies of the nucleic acid sequence. In some aspects, the synthetic enhancer comprises a nucleotide sequence that hybridizes to SEQ ID NO:46 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the synthetic enhancer comprises SEQ ID NO: 46. In some aspects, the synthesis enhancer is integrated 5' to the chicken β -actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 47 is integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest. In some aspects, a chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 47 is integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a CMV enhancer 5' to the promoter in the expression cassette. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% from SEQ ID NO. 12. In some aspects, the CMV enhancer is integrated 3' to the synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO. 12. In some aspects, the CMV enhancer is integrated at the 3 'end of multiple contiguous copies of the synthetic enhancer, e.g., the 3' end of 1, 2, 3, 4, 5 or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated 3' to 3 consecutive copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated with SEQ ID NO:46 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the CMV enhancer is integrated in SEQ ID NO:46, and a nucleic acid sequence 3' end thereof. In some aspects, the CMV promoter is integrated 3 'of the CMV enhancer and 5' of the nucleic acid sequence of interest.
In some aspects, the bacterial-free sequence vector comprises a sequence identical to SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID NO:38 or SEQ ID NO:39, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, said nucleic acid sequence being located 5' to the nucleic acid sequence of interest. In some aspects, the bacterial-free sequence vector comprises the nucleic acid sequence of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 or SEQ ID NO. 39 5' of the nucleic acid sequence of interest. In some aspects, a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, or a nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, comprises all regulatory elements of the expression cassette that are located 5' to the nucleic acid sequence of interest.
In some aspects, the bacterial-free sequence vector comprises a 5' utr comprising an intron, wherein the 5' utr (i.e., the 5' utr comprising the intron) is integrated in the expression cassette between the promoter and the nucleic acid sequence of interest.
In some aspects, the 5'utr can be used to improve splicing and translation of a transgenic transcript from a bacterial-free sequence vector as compared to the same bacterial-free sequence vector lacking the 5' utr.
In some aspects, the intron comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the intron comprises SEQ ID NO:1, and a nucleic acid sequence of seq id no.
In some aspects, the 5' UTR comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 2. In some aspects, the 5' utr comprises SEQ ID NO:2, and a nucleic acid sequence of seq id no.
In some aspects, the 5' UTR comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 4. In some aspects, the 5' utr comprises SEQ ID NO: 4.
In some aspects, the 5' utr further comprises a non-coding sequence integrated in an intron.
In some aspects, the intron is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% >, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID No. 1, or comprises SEQ ID No. 1, and the non-coding sequence is integrated between two nucleotides of the intron, wherein the two nucleotides correspond to SEQ ID NO:1 to any two nucleotides from position 25 to 55.
In some aspects, the non-coding sequence is non-prokaryotic and non-viral. In some aspects, the non-coding sequence is a eukaryotic sequence. In some aspects, the non-coding sequence comprises an intron, UCOE, S/MAR, SV40 enhancer sequence (e.g., one or more SV40 enhancer sequences, such as two, three, four, five or more SV40 enhancer sequences), vertebrate chromatin insulator (e.g., cHS 4), WPRE, or any combination thereof.
In some aspects, the non-coding sequence comprises an S/MAR. In some aspects, the S/MAR is MAR-5, provided herein as SEQ ID NO 9.
In some aspects, the 5' UTR comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 3. In some aspects, the 5' utr comprises SEQ ID NO:3.
in some aspects, the 5' UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 5. In some aspects, the 5' utr comprises SEQ ID NO:5.
in some aspects, the 5' utr is integrated in the expression cassette between the chicken β -actin promoter and the nucleic acid sequence of interest.
In some aspects, the 5' utr is integrated in the expression cassette between the CMV promoter and the nucleic acid sequence of interest.
In some aspects, the 5'utr is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, wherein the promoter is integrated 3' to the CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3' end of the synthetic enhancer comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 12. In some aspects, the CMV enhancer is integrated 3' to the synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO. 12. In some aspects, the CMV enhancer is integrated at the 3 'end of multiple contiguous copies of the synthetic enhancer, e.g., the 3' end of 1, 2, 3, 4, 5 or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated 3' to 3 consecutive copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated with SEQ ID NO:46 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the CMV enhancer is integrated in SEQ ID NO:46, and a nucleic acid sequence 3' end thereof.
In some aspects, the bacterial sequence-free vector comprises a polyadenylation signal integrated 3' to the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a xenopus beta-globin polyadenylation signal, a human beta-globin polyadenylation signal, or a hybrid xenopus and human beta-globin polyadenylation signal. In some aspects, the polyadenylation signal comprises multiple copies of the xenopus laevis beta-globin polyadenylation signal, the human beta-globin polyadenylation signal, or a hybrid xenopus and human beta-globin polyadenylation signal, e.g., 1, 2, 3, 4, or 5 copies. In some aspects, the polyadenylation signal comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15. In some aspects, the polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO. 13, SEQ ID NO. 14 or SEQ ID NO. 15. In some aspects, the polyadenylation tail (i.e., poly (a) tail) is located 3' of the polyadenylation signal. In some aspects, the poly (a) tail is 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or more residues in length. In some aspects, the sequence comprising a polyadenylation signal and a poly (A) tail is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 18. In some aspects, the sequence comprising a polyadenylation signal and a poly (A) tail comprises SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18.
In some aspects, the abacterial sequence vector comprises a vertebrate chromatin insulator in an expression cassette. In some aspects, the vertebrate chromatin insulator is cHS4. In some aspects, a vertebrate chromatin insulator is integrated in an expression cassette between a nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the vertebrate chromatin insulator is integrated in an intron of a 5' utr as described herein.
In some aspects, the vertebrate chromatin insulator comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID No. 8. In some aspects, the vertebrate chromatin insulator comprises SEQ ID NO:8.
in some aspects, the vertebrate chromatin insulator can be used to improve the establishment of a bacterial-free sequence vector (i.e., transfection efficiency) as compared to the same bacterial-free sequence vector without the vertebrate chromatin insulator.
In some aspects, the bacterial sequence-free vector comprises WPRE in the expression cassette. In some aspects, the WPRE is integrated in an expression cassette between a nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the WPRE is integrated in the expression cassette at the 3 'end of the S/MAR described herein and at the 5' end of the polyadenylation signal described herein. In some aspects, the WPRE is integrated in an intron of a 5' utr as described herein.
In some aspects, the WPRE comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 11. In some aspects, WPRE comprises SEQ ID NO:11.
in some aspects, WPRE improves expression of a transgene from a bacterial-free sequence vector compared to the same bacterial-free sequence vector lacking WPRE.
In some aspects, the bacterial sequence-free vector comprises an S/MAR in the expression cassette. In some aspects, the S/MAR is integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal. In some aspects, the S/MAR integration is located in the expression cassette at the 3 'end of the nucleic acid sequence of interest and at the 5' end of the WPRE as described herein. In some aspects, the S/MAR is integrated in an intron of the 5' utr as described herein.
In some aspects, the S/MAR is MAR-3, MAR-4 or MAR-5. In some aspects, the S/MAR comprises a sequence identical to SEQ ID NO:9, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the S/MAR comprises SEQ ID NO 9.
In some aspects, the S/MAR is a human CSP-B MAR or CSP-C MAR. In some aspects, the S/MAR comprises a sequence identical to SEQ ID NO:10 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the S/MAR comprises SEQ ID NO 10.
In some aspects, the S/MAR can be used to improve the expression level, stability, and/or persistence of a bacterial-free sequence vector (e.g., by episomal maintenance and replication, such as expansion of the vector and partitioning into daughter cells, and/or by preventing epigenetic silencing) as compared to the same bacterial-free sequence vector lacking the S/MAR.
In some aspects, the bacterial sequence-free vector comprising any one or more of (b) (i) - (b) (v) as described above (i.e., without DTS) further comprises an enhancer sequence flanking each side of the expression cassette. In some aspects, the enhancer sequences flanking each side of the expression cassette are at least two enhancer sequences flanking each side of the expression cassette. In some aspects, the enhancer sequence is an SV40 enhancer sequence.
In some aspects, the bacteria-free sequence vector comprises DTS. In some aspects, the DTS is located 5' to the expression cassette. In some aspects, the DTS is an SV40 enhancer sequence. In some aspects, the DTS is cell specific. In some aspects, the DTS is specific for smooth muscle cells, embryonic stem cells, type II lung cells, endothelial cells, or osteoblasts.
In some aspects, the bacterial sequence-free vectors described herein further comprise UCOE in the expression cassette. In some aspects, the UCOE is located 5' of the promoter or any enhancer in the expression cassette. In some aspects, the UCOE is integrated in an intron of a 5' utr as described herein.
In some aspects, the UCOE is an A2UCOE. In some aspects, the UCOE comprises a nucleotide sequence that hybridizes to SEQ ID NO:6, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the UCOE is SEQ ID NO:6.
in some aspects, the UCOE is an SRF-UCOE. In some aspects, the UCOE comprises a nucleotide sequence that hybridizes to SEQ ID NO:7 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the UCOE is SEQ ID NO:7.
in some aspects, the UCOE improves expression of the transgene from the bacterial sequence-free vector as compared to the same bacterial sequence-free vector lacking the UCOE.
In some aspects, the bacterial sequence-free vector comprises an enhancer-1 in the expression cassette. In some aspects, enhancer-1 is integrated in the expression cassette 5' of the promoter or any other enhancer. In some aspects, enhancer-1 is integrated between the 3 'end of the UCOE and the 5' end of the CMV enhancer. In some aspects, enhancer-1 comprises a nucleotide sequence that hybridizes to SEQ ID NO:12 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, enhancer-1 is SEQ ID NO:12.
in some aspects, the bacterial sequence-free vector comprises a CMV, EFl, SV, CAG, rho, VDM2, HCR or HLP promoter, or variant thereof, in an expression cassette. In some aspects, the bacterial sequence-free vector comprises a CMV promoter variant in the expression cassette.
In some aspects, the bacterial sequence-free vector comprises the EFl- α promoter in an expression cassette. In some aspects, the bacterial sequence-free vector comprises a CMV enhancer and an EF 1-a promoter in the expression cassette.
In some aspects, the bacterial-free sequence vector comprises a 3'utr in the expression cassette, the 3' utr comprising two copies of the β -globin polyadenylation signal. In some aspects, the 3'utr is integrated 3' of the nucleic acid sequence of interest.
In some aspects, the 3' utr comprises two copies of a xenopus laevis β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:13 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:13.
in some aspects, the 3' utr comprises two copies of a human β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:14, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:14.
in some aspects, the 3' utr comprises one copy of a xenopus laevis β -globin polyadenylation signal and one copy of a human β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:15, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:15.
In some aspects, the 3' utr further comprises a poly (a) tail comprising 100 to 120 adenine nucleotides, i.e., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 adenine nucleotides.
In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:16 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:16.
in some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:17 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:17.
in some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:18, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:18.
The nucleic acid sequence of interest in a bacterial sequence-free vector as described herein includes any nucleic acid sequence described herein when used to produce an expression vector for a bacterial sequence-free vector.
In some aspects, the bacterial sequence-free vectors described herein comprise Cas endonuclease target sequences located 5 'and 3' of the nucleic acid sequence of interest (i.e., sequences homologous to the gRNA targeting sequence), wherein the target site for gene editing (e.g., the target site in the chromosome) comprises the same Cas endonuclease target sequence.
In some aspects, the bacterial sequence-free vectors described herein comprise a CRISPR-Cas system. In some aspects, the bacteria-free sequence vector comprises a tRNA-gRNA polycistronic flanking each side of the sequence encoding a Cas endonuclease (e.g., immunosilent Cas9- β2). In some aspects, the bacterial-free sequence vector comprises a 5'utr (e.g., 5' utr 1) as described herein, comprising a tRNA-gRNA polycistron in an intron. In some aspects, the bacterial-free sequence vector comprises a chimeric intron as described herein comprising a tRNA-gRNA polycistron. In some aspects, the EF 1-alpha promoter described herein comprises a tRNA-gRNA polycistronic in the inherent intron. In some aspects, the polyadenylation signals or 3' UTRs described herein comprise tRNA-gRNA polycistronic. In some aspects, the nucleic acid sequence of interest and the self-limiting CRISPR-Cas system as described herein are located on a single bacterial sequence-free vector as described herein. In the latter aspect, the sequence comprising the self-limiting CRISPR-Cas system is located 5' to the sequence comprising the nucleic acid sequence of interest flanked by homology arms.
The bacterial sequence-free vectors described herein may comprise any combination of the above modifications. In some aspects, the combination provides a synergistic effect.
In some aspects, the bacterial sequence-free vector is a circular covalently closed vector.
In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
Provided herein are recombinant cells comprising the bacterial no-sequence vectors disclosed herein.
Other expression vectors
The improvements and modifications described above can also be applied to other expression vectors, such as, but not limited to, expression vectors for direct gene expression rather than for the production of vectors without bacterial sequences. In some aspects, the nucleic acid sequences described herein are provided as DNA sequences, and the expression vector is a DNA expression vector. In some aspects, the nucleic acid sequences described herein are provided as RNA sequences, and the expression vector is an RNA expression vector. The RNA sequence may correspond to a DNA sequence provided in any of the SEQ ID NOs herein, or may correspond to a DNA sequence complementary to a DNA sequence provided in any of the SEQ ID NOs herein.
Provided herein are polynucleotides comprising any combination of the nucleic acid sequences described herein.
Provided herein are polynucleotides comprising the following nucleic acid sequences: introns, 5 'utrs containing introns, and/or 3' utrs as described herein.
Provided herein are polynucleotides comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of SEQ ID NOs 1, 2, 3, 5, 13, 14, 15, 16, 17, or 18. In some aspects, the polynucleotide comprises 100 to 120 adenine nucleotides at the 3' end of the nucleic acid sequence. In some aspects, the polynucleotide comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs 13, 14, or 15, and has 100 to 120 adenine nucleotides at the 3' end of the nucleic acid sequence. In some aspects, the polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NOs 1, 2, 3, 5, 13, 14, 15, 16, 17 or 18.
Provided herein are expression vectors comprising one or more polynucleotides described herein. In some aspects, the expression vector comprises a polynucleotide comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of SEQ ID NOs 1, 2, 3, 5, 13, 14, 15, 16, 17, or 18. In some aspects, the polynucleotide comprises 100 to 120 adenine nucleotides at the 3' end of the nucleic acid sequence. In some aspects, the polynucleotide comprises a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs 13, 14, or 15, and has 100 to 120 adenine nucleotides at the 3' end of the nucleic acid sequence. In some aspects, the expression vector comprises a polynucleotide comprising a nucleic acid sequence of any one of SEQ ID NOs 1, 2, 3, 5, 13, 14, 15, 16, 17 or 18. In some aspects, the expression vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID nos. 2, 3, or 5, and (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID nos. 13, 14, 15, 16, 17, or 18, or (b) a nucleic acid sequence comprising SEQ ID NOs: 13. 14 or 15, and a polynucleotide of 100 to 120 adenine nucleotides at the 3' end of the nucleic acid sequence.
Provided herein are expression vectors comprising: a 5'utr comprising an intron, wherein said 5' utr is integrated in the expression cassette between the promoter and the nucleic acid sequence of interest; and/or a 3' utr comprising two copies of a β -globin polyadenylation signal, said 3' utr being integrated 3' of the nucleic acid sequence of interest in the expression cassette.
In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:2, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 5' utr comprises SEQ ID NO:2, and a nucleic acid sequence of seq id no.
In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:4, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 5' utr comprises SEQ ID NO: 4.
In some aspects, the 5' utr further comprises a non-coding sequence integrated in an intron.
In some aspects, the intron is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% >, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID No. 1, or comprises SEQ ID No. 1 and the non-coding sequence is integrated between two nucleotides in the intron, the two nucleotides corresponding to SEQ ID NO:1 to any two nucleotides from position 25 to 55.
In some aspects, the non-coding sequence is non-prokaryotic and non-viral. In some aspects, the non-coding sequence is a eukaryotic sequence. In some aspects, the non-coding sequence comprises an intron, UCOE, S/MAR, SV40 enhancer sequence (e.g., one or more SV40 enhancer sequences, such as two, three, four, five or more SV40 enhancer sequences), vertebrate chromatin insulator (e.g., cHS 4), WPRE, or any combination thereof.
In some aspects, the non-coding sequence is an S/MAR. In some aspects, the S/MAR is MAR-5, provided herein as SEQ ID NO 9.
In some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:3, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 5' utr comprises SEQ ID NO:3.
in some aspects, the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:5 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 5' utr comprises SEQ ID NO:5.
In some aspects, the 3' utr comprises two copies of a xenopus laevis β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:13 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:13.
in some aspects, the 3' utr comprises two copies of a human β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:14, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:14.
in some aspects, the 3' utr comprises one copy of a xenopus laevis β -globin polyadenylation signal and one copy of a human β -globin polyadenylation signal. In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:15, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:15.
In some aspects, the 3' utr further comprises a poly (a) tail comprising 100 to 120 adenine nucleotides, i.e., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 adenine nucleotides.
In some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:16 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:16.
in some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:17 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:17.
in some aspects, the 3' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:18, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the 3' utr is SEQ ID NO:18.
Provided herein are expression vectors comprising a synthetic enhancer comprising a nucleotide sequence that hybridizes to SEQ ID NO:12 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the expression vector comprises a synthetic enhancer comprising the amino acid sequence of SEQ ID NO: 12. In some aspects, the synthesis enhancer comprises multiple contiguous copies of the nucleic acid sequence, e.g., 1, 2, 3, 4, 5, or more contiguous copies. In some aspects, the synthesis enhancer comprises 3 consecutive copies of the nucleic acid sequence. In some aspects, the synthetic enhancer comprises a nucleotide sequence that hybridizes to SEQ ID NO:46 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the synthetic enhancer comprises SEQ ID NO: 46.
Provided herein are expression vectors comprising a nucleotide sequence that hybridizes to SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO:39, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical. In some aspects, the expression vector comprises the nucleic acid sequence of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 or SEQ ID NO. 39. In some aspects, a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39, or a nucleic acid sequence of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39, comprises all regulatory elements of the expression cassette located 5' of the nucleic acid sequence of interest of the expression vector.
V. composition
Provided herein are compositions comprising the expression vectors or bacterial sequence-free vectors described herein.
Various methods are known in the art and are suitable for introducing nucleic acids into cells. Examples include, but are not limited to, electroporation, calcium phosphate mediated transfer, nuclear transfection, sonoporation (sonoporation), heat shock, magnetic transfection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG), etc.), or cell fusion.
Nanoparticle carriers such as liposomes, micelles, and polymeric nanoparticles have been investigated to improve the bioavailability and pharmacokinetic properties of therapeutic agents by various mechanisms, such as Enhanced Permeability and Retention (EPR) effects.
Other improvements may be achieved by conjugating targeting ligands to nanoparticles to achieve selective delivery to target cells. For example, receptor-targeted nanoparticle delivery has been demonstrated to improve therapeutic responses in vitro and in vivo. Targeting ligands that have been investigated include folic acid, transferrin, antibodies, peptides and aptamers. In addition, various functions may be incorporated into the design of the nanoparticle, for example, to allow imaging and triggering intracellular drug release.
In some aspects, the compositions of the present invention further comprise a delivery agent. In some aspects, the delivery agent is a nanoparticle. In some aspects, the delivery agent is selected from the group consisting of liposomes, non-lipopolymer molecules, endosomes (endosomes), and any combination thereof.
In some aspects, the delivery agent (e.g., nanoparticle) comprises a targeting ligand.
In some aspects, the composition further comprises a physiologically acceptable carrier, excipient, or stabilizer. See, e.g., remington, the Science and Practice of Pharmacy,22 nd ed. (2013). Acceptable carriers, excipients, or stabilizers may include those that are non-toxic to the individual. In some aspects, the composition or one or more components of the composition are sterile. The sterile components may be prepared, for example, by filtration (e.g., through a sterile filtration membrane) or by radiation (e.g., by gamma radiation).
In some aspects, the composition comprising an expression vector or a bacterial sequence-free vector as described herein is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
When added to pharmaceutical compositions, the excipients of the present invention may be described as "pharmaceutically acceptable" excipients, meaning that the excipients are compounds, materials, compositions, salts and/or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals and that do not cause excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. In some aspects, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or recorded in the U.S. pharmacopeia or other generally recognized international pharmacopeia for use in animals, and more particularly in humans. Various excipients may be used. In some aspects, a vehicle The shaping agent may be, but is not limited to, an alkaline agent, a stabilizer, an antioxidant, a binder, a release agent, a coating agent, an external phase component, a controlled release component, a solvent, a surfactant, a wetting agent, a buffer, a filler, a lubricant, or a combination thereof. Excipients other than those discussed herein may include, but are not limited to, remington: the Science and Practice of Pharmacy,22 nd ed. (2013). Herein, the inclusion of an excipient into a particular class (e.g., "solvent") is intended to illustrate, but not limit, the effect of the excipient. One particular excipient may belong to multiple categories.
The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Exemplary routes of administration include enteral, topical, parenteral, oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, or intraperitoneal administration, or inhalation. "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intrapleural and intrasternal injection and infusion, and in vivo electroporation. In some aspects, the formulation is administered by a non-parenteral route, in some aspects, by oral administration. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical administration.
In some aspects, the pharmaceutical composition is lyophilized.
VI therapeutic uses and methods
Provided herein is a method of treating a disease or disorder in an individual in need thereof, comprising administering to the individual an expression vector, a bacterial-free sequence vector or a pharmaceutical composition described herein.
The expression vector, bacterial sequence free vector or composition may be administered to a subject by any route of administration effective to treat the disease or disorder.
In some aspects, administration is enteral, topical, parenteral, oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, intrathecal or intraperitoneal administration, inhalation, or cerebrospinal fluid (CSF) -based delivery by Intraventricular (ICV) injection, cerebellar medullary administration (ICM) or lumbar intrathecal puncture (LIT).
In some aspects, the administration is by parenteral or non-parenteral administration.
In some aspects, parenteral administration is by injection or infusion.
In some aspects, parenteral administration is by intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, retroorbital, intracerebroventricular, subarachnoid, intraspinal, epidural, intrapleural or intrasternal injection or infusion, or by in vivo electroporation, nuclear transfection, microbubble or ultrasound administration.
In some aspects, the parenteral administration is oral, topical, epidermal, mucosal, intranasal, vaginal, rectal, or sublingual administration.
In some aspects, administration is by oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, or intraperitoneal administration, or by inhalation.
In some aspects, administration is by oral, nasal, or pulmonary administration. In some aspects, administration is by nasal administration.
The administration may be, for example, one, multiple, and/or over one or more long periods of time. In some aspects, the administration is one, two (e.g., a first administration followed by a second administration after about 1, about 2, about 3, about 4, or more weeks), about weekly, about monthly, about every 2 months, about every 3 months, about every 4 months, about every 6 months, about yearly, or about every ten years.
Provided herein is a method of gene editing comprising inserting a nucleic acid sequence of interest from an expression vector, bacterial no-sequence vector or pharmaceutical composition described herein into a target site of gene editing.
In some aspects, the insertion is performed by non-homologous end joining.
In some aspects, the insertion is by homology directed repair. In some aspects, the nucleic acid sequence of interest is flanked by 5 'and 3' homology arms as described herein.
In some aspects, the nucleic acid sequence of interest is homologous to a target site for gene editing and comprises one or more nucleotide insertions, deletions, inversions, or rearrangements compared to the target site.
In some aspects, the nucleic acid sequence of interest is non-homologous to a target site for gene editing.
In some aspects, the nucleic acid sequence of interest restores the function of a deletion, corrects an abnormality, or provides other functions associated with the target site of gene editing.
In some aspects, the nucleic acid sequence of interest is used to knock out gene expression associated with a target site of gene editing.
In some aspects, the gene editing method is a method of treating a disease or disorder in an individual in need thereof.
In some aspects, the nucleic acid sequence of interest is used for in vivo gene editing.
In some aspects, the nucleic acid sequence of interest is used for in vitro gene editing.
In some aspects, the nucleic acid sequence of interest is used for ex vivo gene editing (e.g., cell therapy, such as CAR T cell therapy).
In some aspects, the method is an in vitro method. In some aspects, the in vitro methods further comprise administering to the cells an expression vector, a bacterial-free sequence vector, or a pharmaceutical composition of the invention (e.g., for in vitro or ex vivo gene editing). In some aspects, the in vitro method further comprises administering to the cell an endonuclease for gene editing, or a genome editing system or component thereof (e.g., cas endonuclease and gRNA for CRISPR-Cas system). In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN or meganuclease gene editing system.
In some aspects, the method is an in vivo method. In some aspects, the in vivo methods further comprise administering to the subject an expression vector, a bacterial sequence-free vector, or a pharmaceutical composition of the invention. In some aspects, the in vivo methods further comprise administering to the individual an endonuclease for gene editing, or a genome editing system or component thereof (e.g., cas endonuclease and gRNA for CRISPR-Cas system). In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN or meganuclease gene editing system.
The endonuclease or genome editing system or components thereof for gene editing may be administered by any method described herein or known in the art for administering a nucleic acid sequence and/or polypeptide to a cell or individual, including by electroporation or a vector suitable for administration. For example, in terms of comprising a CRISPR-Cas system, RNA and/or gRNA encoding Cas may be administered, cas and/or gRNA may be administered directly, a bacterial-free sequence vector or expression vector encoding Cas and/or gRNA described herein may be administered, or any other suitable vector encoding Cas and/or gRNA known in the art may be administered.
In some aspects, the nucleic acid of interest is provided in a linear covalently closed bacterial sequence-free vector (i.e., msDNA) as described herein. In some aspects, the use of the linear covalently closed bacterial-free sequence vector in gene editing can avoid any undesired non-homologous end joining, as the ends of the bacterial-free sequence vector are closed and do not react with double strand breaks. In some aspects, homologous targeted repair can be enhanced using the linear covalently closed bacterial-free sequence vector in gene editing. In some aspects, the recombination rate of homology directed repair is higher when the nucleic acid sequence of interest is provided by a linear covalently closed bacterial sequence-free vector as described herein than when the nucleic acid sequence of interest is provided by a circular supercoiled vector.
The following examples are provided for illustration and not limitation.
Examples
Example 1-expression vectors containing chimeric introns or 5' UTRs
A. Expression vector
The eGFP coding sequences in the parental ministring expression vector pGL 2-SS-CAG-eGFP-BGpA-SS (Mediphage Bioceuticals, inc., toronto, CA, U.S. patent Nos.9,290,778, and 9,862,954) were replaced with those encoding enhanced green fluorescent protein (eGFP) and modified Expression cassettes for luciferase reporter molecules so that they are located between two specific super-sequence (SS) sites of the parent vector, thereby preparing a polygenic expression vector, wherein said +.>The luciferase reporter is modified to have a secretion sequence for extracellular expression (NLuc, promega Corporation).
The parental and polygenic vectors contain the CAG promoter, which is a synthetic promoter comprising the Cytomegalovirus (CMV) enhancer, the promoter from chicken β -actin, and the chimeric intron.
The map of the polygene expression vector is shown in fig. 1 (pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS) comprising specific super Sequence Sites (SS) with recombinase target sequences (telL, FRT (minimal) and loxP) flanking the polygene expression cassette comprising CAG promoter, sequences encoding enhanced green fluorescent protein (eGFP) and secreted nano luciferase (SecNLuc) linked by P2A and T2A self-cleaving peptides (SecNLuc-2A-eGFP), and rabbit β -globin polyadenylation signal (BGpA). The nucleic acid sequence of the vector is represented by SEQ ID NO: 19.
A second polygenic expression vector was prepared by cloning the same eGFP and Nluc sequences together with the 5' UTR into the pcDNA3.1 vector (Thermo Fisher Scientific). The map of the expression vector is shown in FIG. 2 (vector pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA), and the expression vector comprises a multigene expression cassette containing a CMV enhancer/promoter, sequences encoding eGFP and SecNLuc linked by a P2A self-cleaving peptide (SecNLuc-P2A-eGFP) and bovine growth hormone polyadenylation signal (bGHPA). The nucleic acid sequence of the vector is represented by SEQ ID NO: 20.
B. Transfection of HEK293 cells
Adherent human embryonic kidney 293 (HEK 293) cells were grown at 1X10 5 Individual cells/well were seeded into 24-well plates.
Complexes of expression vectors (1 μg) and lipofectamine (3 μl) were prepared and incubated for (1) pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS, (2) pcDNA-CMV-5' utr-SecNLuc-P2A-eGFP-bGHpA, and (3) pGL 2-SS-CAG-eGFP-BGpA-SS, respectively, using standard protocols.
HEK293 cells in wells were transfected separately by electroporation using these three complexes and then incubated for 48 hours. HEK293 cells in other wells were treated with 3 μl of lipofectamine without plasmid as negative control.
Cells were assessed 48 hours post-transfection for cytoplasmic GFP expression and luciferase expression.
C. Cytoplasmic GFP expression
Cytoplasmic GFP expression was used as a measure of transfection efficiency and gene expression level of the multigenic expression vector. Expression was assessed by fluorescence microscopy and the average GFP expression/intensity of the experimental expression vectors (pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS) and pcDNA-CMV-5' utr-SecNLuc-P2A-eGFP-bGHpA) was measured relative to negative control (cells treated with plasmid-free lipofectamine) and positive control (pGL 2-SS-CAG-eGFP-SS) (also referred to herein as parent plasmid CAG-GFP, i.e. PP-CAG-GFP).
Live cell imaging of fluorescent cells in auto-exposure mode showed that experimental expression vectors produced GFP in which pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS with chimeric intron and pcDNA-CMV-5'utr-SecNLuc-P2A-eGFP-bGHpA with 5' utr had similar expression. See fig. 3 and 4. Based on the average relative fluorescence intensity, the polygenic expression of the experimental expression vector did not affect GFP expression compared to the positive control. As above (id.). The average fluorescence intensity in cells transfected with the experimental expression vector was at least 3-fold higher than that of negative control cells. See fig. 4.
D. Luciferase expression
UsingLuciferase assay System (Promega), according to the manufacturer's protocol, the intensity of secreted luciferase in the medium of transfected cells and negative control cells was measured to assess luciferase expression. Both experimental expression vectors expressed luciferase. See fig. 5. The average relative luciferase intensity in the medium of cells transfected with the experimental expression vector was at least 300-fold higher than in the medium of negative control cells. Same as above (Id.)
Example 2 expression vectors containing WPRE and engineered 5' UTR
A. Expression vector
A polygenic expression vector was prepared by cloning a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) between the sequence encoding eGFP and BGpA in the expression vector of fig. 1. The map of the obtained expression vector is shown in FIG. 6 (pGL 2-SS. Times. -CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS. Times.). The nucleic acid sequence of the vector is provided as SEQ ID NO:21.
Another polygenic expression vector was prepared containing a CMV enhancer/promoter and an engineered 5' utr in place of the CAG promoter in fig. 6, wherein the engineered 5' utr contains an internal minimal intron sequence (i.e., 5'UTR1,SEQ ID NO:2). The map of the obtained expression vector is shown in FIG. 7 (pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS). The nucleic acid sequence of the vector is provided as SEQ ID NO:22.
another polygenic expression vector was prepared containing a CMV enhancer/promoter and an engineered 5' utr instead of the CAG promoter in fig. 6, wherein the engineered 5' utr contains an intron (i.e., 5'UTR2,SEQ ID NO:5) into which MAR-5 is integrated. The map of the obtained expression vector is shown in FIG. 8 (pGL 2-SS-CMV-UTR 2-SecNLuc-2A-eGFP-WPRE-BGpA-SS). The nucleic acid sequence of the vector is provided as SEQ ID NO:23.
B. luciferase expression levels and persistence
Digestion of adherent HEK293 cells dispersed in electroporation medium at 1X10 6 Individual cells/tubes were counted.
Expression vectors (1 μg) were prepared and incubated with cells using standard procedures for (1) pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS, (2) pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS, (3) pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS, and (4) pGL 2-SS-CMV-UTR 2-SecNLuc-2A-eGFP-WPRE-BGpA-SS, respectively.
HEK293 cells electroporated with the puc57 plasmid lacking the mammalian expression cassette served as negative controls.
Following electroporation, HEK293 cells were grown at 3x10 5 Individual cells/wells are seeded and attached within the wells.
Nano-bolls were used for each of the 4 transfections and negative controls on days 2, 6, 10, 14, 17, 20, 27 and 34 post electroporationLuciferase assay System (Promega), luciferase expression was assessed by measuring the intensity of secreted luciferase in 20. Mu.L of cell culture medium in triplicate according to the manufacturer's protocol. Use->The enzyme-labeled instrument measures luciferase activity and is shown in Relative Luminometer Units (RLU). Statistical analysis of luciferase activity was performed by Student T-test. Referring to fig. 9, the expression levels in the medium of cells transfected with pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS (pGL 2-SecNLuc-eGFP), pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS (WPRE), pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-BGpA-SS (5 'utr1+wpre) or pGL 2-SS-CMV-UTR 2-SecNLuc-2A-eGFP-BGpA-SS (5' utr2+wpre) relative to the negative control (neg.ctl). * =p<0.05,**＝p<0.01,***＝p<0.001 and =p<0.0001。
Luciferase expression was detected from cells transfected with any of the four expression vectors throughout the experiment. pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS, pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS and pGL 2-SS-CMV-UTR 2-SecNLuc-2A-eGFP-WPRE-BGpA-SS each show significantly higher luciferase expression, with pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS showing the highest expression enhancement.
C. Amplification of the vector to daughter cells and luciferase expression
HEK293 cells were transfected with four expression vectors or the puc57 plasmid (as negative control) as described in example B. Cells were passaged five times per week. At the time of cell passage, the cells were re-seeded at 1/6 of the original cell density for the 1 st to 3 rd passages of cells; for the 4 th-5 th generation cells, the cells were re-seeded at 1/10 of the original cell density. For each cell passage, secreted luciferase expression was measured 6-8 days after cell re-inoculation as described in section B of this example. Referring to fig. 10, the expression levels in the medium of vector transfected cells compared to the negative control at each generation are shown. Statistical analysis and p-values were as described in example B.
At each generation, luciferase expression was detected from cells transfected with any one of the four expression vectors, indicating that the vector was transmitted to daughter cells with persistent luciferase expression. pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS, pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS, and pGL 2-SS-CMV-UTR 2-SecNLuc-2A-eGFP-WPRE-BGpA-SS show significantly higher luciferase expression per generation, with pGL 2-SS-CMV-UTR 1-SecNL-2A-eGFP-RE-BGpA-SS showing the highest enhancement of expression.
In subsequent studies, it was also observed that msDNA amplified to daughter cells with persistent luciferase expression.
Briefly, using the methods described herein and in U.S. patent nos. 9,290,778 and 9,862,954, msDNA was generated from pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS in an inducible escherichia coli vector production system. Complexes with lipofectamine were prepared using (1) msDNA (i.e. msDNA-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA) (2) parental plasmid (i.e. pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS) and (3) conventional plasmid with luciferase expression cassette (i.e. pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHpA), respectively. HEK293 cells in wells were transfected with 0.25pmol total of vector/well by electroporation, respectively. Cells were passaged 7 times, with 10-fold dilution of cells per passage. Relative luciferase intensities at passage 1, 2, 3, 4, 5, 6 and 7 were measured on days 8, 15, 24, 31, 38, 45 and 52 respectively.
As shown in fig. 11, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS (i.e., pDNA (cmv+u1+w)) and msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (i.e., msDNA (cmv+u1+w)) exhibited much higher levels of persistent transgene expression than pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHpA (i.e., conventional plasmid without supersequence (conventional pcDNA)) in all generations. Compared to the dataset of conventional plasmids, p <0.01, =p <0.001 and p <0.0001.
D. Vector expansion to daughter cells and eGFP expression
Cells were transfected and passaged with pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS (pGL 2-SecNLuc-eGFP) or pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS (5' utr1+wpre) according to example C, and analyzed for eGFP expression.
For each generation, imaging was performed 6 to 8 days after passage of the cells. UsingCytation TM The plate reader was used for live cell imaging. Referring to FIG. 12A, representative photomicrographs of fluorescence in HEK-293 cells of passages 1, 2, 3 and 5 are shown. eGFP expression was detected in cells transfected with either expression vector at each generation, indicating that these vectors have been transferred to daughter cells with persistent expression of eGFP. pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS showed stronger fluorescent signals per generation compared to pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS, indicating higher transfection efficiency.
For each expression vector, a representative image was taken from triplicate wells and the number of eGFP expressing cells (gfp+) was quantified by manual cell counting using ImageJ computer software. Gfp+ cells were statistically analyzed using Student t-test. Referring to fig. 12B, a line graph of gfp+ cells observed from the field of live cell fluorescence images in triplicate at each generation is shown (the difference in triplicate images is not significant (ns) =p > 0.05).
Using ImageJ software, each gfp+ cell was manually selected and the Mean Fluorescence Intensity (MFI) of each cell was measured based on pixel intensity. To calculate the final MFI value for each cell, the following formula was used (final MFI = cell MFI-background MFI). MFI measurements of at least 50 cells were obtained from each of the 3 images acquired for each treatment group. All MFI measurements are then aggregated and used to generate a point map. Statistical analysis was performed using Student t-test. Referring to fig. 12C, a MFI plot of passage 5 is shown, wherein pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS n=257 cells, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS n=414 cells, =p <0.0001. The bar graph in fig. 12C shows the average MFI values of all gfp+ cells measured. The MFI of pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS was measured to be about 3 times higher than pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS.
EXAMPLE 4 non-viral delivery Using msDNA in animal models
Studies were performed to assess targeted delivery of msDNA to the liver, retina and brain. For each target tissue, different routes of administration (ROA), dosages, dosing regimens, and delivery techniques were evaluated. The kinetics of secreted luciferase expression, cytoplasmic eGFP expression levels, and Transfection Efficiency (TE) were evaluated. Furthermore, tolerance to msDNA was assessed after single or multiple injections by physiological assessment, histomorphometric analysis, plasma cytokine assay and hepatotoxicity analysis.
In all delivery techniques, msDNA shows strong efficacy and tolerability properties in brain and liver tissue by multiple Intraventricular (ICV) or Hydrodynamic (HDI) and Intravenous (IV) injections. Adult mice treated with msDNA showed sustained secretion of luciferase levels following a single IV injection [ ] after IV injection>10 8 RLU/mg protein). msDNA showed a long lasting @ in liver tissue following a single IV injection>100 days) expression. Significant biodistribution to deep tissue regions was also demonstrated, with 80% to 97% of TE in the brainstem, cerebellum, cortex and thalamus. Three ICV injections using nanocarrier-msDNA complexesThe injection did not show any side effects.
A. Liver
1. Single high dose 2mg/kg (50 μg) hydrodynamic injection of luciferase expression from carrier-free naked plasmid
C57BL/6J male wild-type adult 8-12 week old mice received a single high dose (50 μg) of carrier-free pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control without super sequence), pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS, or pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS by tail vein hydrodynamic injection (HDI). Plasma from treated mice was collected at days 1, 3, 7, 10, 15, 22, 28, 42 and 56 post HDI to examine luciferase gene expression. On day 1 after vector administration, all mice showed high levels of luciferase expression (10 per mg of plasma protein 8 -10 9 RLU). On day 7, pCAGLuc and pCAGLuc-WPRE treated mice produced 10 7 -10 8 RLU/mg plasma protein, but pGSNLuc-WPRE treated mice produced lower luciferase levels (about 10 6 RLU/mg protein). All mice showed low levels of luciferase expression after 8 weeks of vector administration (about 10 5 RLU/mg protein). The rapid decrease in luciferase levels may be due to humoral or cell-mediated immune responses induced in plasmid-treated mice. See fig. 13-14.
2. Single low dose 0.2mg/kg (5 μg) hydrodynamic injection of luciferase expression from carrier-free naked plasmid
To test the dose response of plasmid DNA after non-viral gene delivery in animal models, single low doses of 0.2mg/kg (5 μg) of pcDNA-CMV-5' utr-SecNLuc-P2A-eGFP-bGHpA (positive control without super sequence, 2 mice), pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS (2 mice) or pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS (2 mice) were administered via tail vein HDI to C57BL/6J male adult 8-12 week old mice. Another 2 mice were not injected and served as negative controls. Mouse plasma was collected 1, 3, 7, 10, 15, 22, 28, 42 and 56 days after HDI to examine luciferase gene expression Reaching the end of the process. Mice treated with pGL 2-SS-CAG-SecNLuc-2A-eGFP-BGpA-SS and pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS showed sustained high levels of luciferase expression for more than 8 weeks after vector administration (10 7 -10 8 RLU/mg protein) and the expression level was 100-fold higher than that of a conventional control plasmid having an isogenic expression cassette but no Super Sequence (SS). See fig. 15.
In vivo whole body bioluminescence imaging (BLI) was performed using IVIS by intraperitoneal injection of fluorofurimazine (FFz) at 1:5 dilution 24 hours after HDI vehicle. The BLI was shown to correlate with luciferase levels in plasma samples (data not shown).
3. Single low dose 0.2mg/kg (5 μg) hydrodynamic injection of luciferase expression of naked msDNA without carrier
In the inducible E.coli vector production system, msDNA was generated from pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS and pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS using the methods described herein and in U.S. Pat. Nos. 9,290,778 and 9,862,954.
Single low doses of 0.2mg/kg (5 μg) of carrier-free pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control, 5 mice), msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (5 mice) or msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (5 mice) were administered to C57BL/6J male wild type adult 8-12 week old mice by tail vein hydrodynamic injection (HDI). Another 2 mice were not injected and served as negative controls. Plasma from treated mice was collected at days 1, 3, 7, 10, 15, 22, 28, 42 and 56 post HDI to examine luciferase gene expression.
Like plasmid-treated mice, msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-treated mice produced sustained high levels of luciferase expression for more than 8 weeks after vector administration (10 7 -10 8 RLU/mg protein), whereas luciferase expression in msDNA-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-treated mice decreased to low levels (10) in less than one month 6 RLU/mg protein). The rapid decrease in luciferase expression in msDNA-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-treated mice may be due to CMV promoter in hepatocytesSilencing of the mover.
Luciferase gene expression was confirmed by whole body in vivo imaging using IVIS.
Table 1 below provides data from individual mice on days 1, 7 and 28, including luciferase expression data (RLU/mg protein) in plasma samples and data detected by BLI (photons/sec).
TABLE 1
As shown in fig. 16, mice treated with msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS showed a 10-fold increase in luciferase expression compared to the parental plasmid pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA at day 56 post HDI.
This data demonstrates that non-viral delivery in mice with msDNA is highly efficient and that the stability of the resulting gene expression exceeds two months.
4. eGFP expression of single low dose 0.2mg/kg (5 μg) hydrodynamic injection of vehicle-free naked msDNA
Intracellular cytoplasmic eGFP expression levels were assessed by ELISA. Briefly, liver samples were collected from mice on day 56 after HDI using a single low dose of 0.2mg/kg (5 μg) carrier and homogenized to extract proteins as described in section 3. Total protein concentration was determined from liver lysates. GFP protein levels were then analyzed by ELISA.
By comparing the data in fig. 17 with the luciferase data, it is evident that cytoplasmic GFP expression levels are directly related to the secretion levels of luciferase from the same construct.
As shown in FIG. 18, single HDI tail intravenous injection of 5 μg of carrier-free msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BgpA showed strong and durable cytoplasmic eGFP expression in liver tissue for a minimum of 56 days after HDI.
5. Expression and tolerability of msDNA in liver after a single intravenous injection at low doses
0.3mg/kg of pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA (positive control without super sequence), msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA, msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS, or pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS were administered to C57BL/6J male adult 8-12 week old mice by single intravenous tail vein injection. The carrier (carrier) also served as a negative carrier control.
In vivo whole body bioluminescence imaging (BLI) was performed using IVIS as described above on days 1, 3, 10, 30, 58, 92, 119 and 174 following a single IV injection of vector. As shown in FIG. 19, the msDNA construct is superior to the precursor and conventional plasmids, showing high and durable luciferase secretion.
Serum alanine Aminotransferase (ALT) levels, hepatotoxicity and cytokine responses were also assessed following vector injection. The precursor plasmid and msDNA containing the CAG promoter showed higher tolerance compared to the construct containing the CMV promoter. However, msDNA containing CMV promoter showed significantly lower cytokine and hepatotoxic responses compared to CMV precursor parents and conventional plasmids. See below
Table 2 shows the cytokine concentration (pg/mL) and the enzyme concentration (U/L) of liver function markers at 4 hours and 14 days after injection.
B. Brain
msDNA was generated from pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS and pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS in an inducible escherichia coli vector production system using the methods described herein and in U.S. patent nos. 9,290,778 and 9,862,954.
3 intra-brain (ICV) injections of 1 μg of DNA each were made on days 0, 14 and 28 post-implantation through the implanted cannulas, and adult wild-type mice were administered either with nano-vehicle-formulated msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (3 mice) or nano-vehicle-formulated msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (3 mice). Animals were euthanized on day 42 post-implantation and sagittal brain sections were collected from cortex, thalamus, brainstem and cerebellum.
FIG. 20 shows cortical, thalamus, brainstem and cerebellar sections from mice #1 from the treatment group injected with msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA. Transfection efficiencies of msDNA in sections were determined to be 81.9%, 73.0%, 69.2% and 96.0% in cortex, thalamus, brainstem and cerebellum (purkinje cells), respectively. Transfection efficiency was calculated as the percentage of cells positive for both GFP and DAPI among all DAPI positive cells.
GFP expression was compared in cortex, thalamus and brainstem sections of mice #1 in the treatment group injected with msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA and mice injected with the control plasmid pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHPA, showing that the transfection efficiency and thus the GFP expression was higher compared to this conventional plasmid (data not shown).
Sections of cortex and thalamus from mice #2 of the msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA injected treatment group are shown in fig. 21, and sections of brainstem and cerebellum are shown in fig. 22. Neurons were labeled with the neuron marker NeuN and transfected cells were shown to express GFP. Transfection efficiencies in cortex, thalamus, brain stem and cerebellum (purkinje cells) were determined to be 99.6%, 98.8%, 98.5% and 80.8%, respectively. Transfection efficiency was calculated as the percentage of cells positive for both GFP and NeuN among all NeuN positive cells.
Sections of cortex and thalamus from mice #1 from the treatment group injected with msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA are shown in fig. 23, and sections of brainstem and cerebellum are shown in fig. 24. Neurons were labeled with the neuron marker NeuN and transfected cells were shown to express GFP. Transfection efficiencies in cortex, thalamus, brainstem and cerebellum (purkinje cells) were determined to be 91.1%, 88.8%, 73.7% and 92.1%, respectively. Transfection efficiency was calculated as the percentage of cells positive for both GFP and NeuN among all NeuN positive cells.
Table 3 below summarizes the transfection efficiencies of the mice injected with either msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA ("CAG-WPRE") or msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA ("CMV-WPRE") discussed above.
Table 3.
Repetition of ICV injections by implanted cannulas resulted in good overall tissue integrity with no signs of cytotoxicity or neurodegeneration.
The data show that msDNA is reproducible and results in high transfection efficiency, biodistribution and transgene expression in multiple brain regions without morphological side effects.
EXAMPLE 5 efficacy and safety in human cells
pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHpA (positive control), msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA, pGL 2-SS-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-BGpA-SS, msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA, or pGL 2-SS-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS, respectively, are lipid complexed with lipid nanoparticle carriers.
Human T cells (Pan-T (TA+)) and hepatocytes (Huh 7) were transfected with the lipid-complexed vector at doses of 0.3. Mu.g/mL or 1. Mu.g/mL.
Lipid-complexed msDNA vectors showed high expression in both cell types on days 3 and 5 post-transfection compared to the parental and conventional plasmids. See fig. 25-26.
Lipid-complexed msDNA is also well tolerated in isolated human Peripheral Blood Mononuclear Cells (PBMCs). In particular, msDNA showed significantly lower cytokine profile levels in human PBMC compared to conventional plasmids (data not shown).
EXAMPLE 6 homologous directed repair Using msDNA
Studies were performed to evaluate msDNA-mediated homology-directed repair compared to conventional plasmid DNA.
Conventional plasmids (plasmid DNA HDR-GOI-HDR) were generated using expression cassettes containing genes of interest (GOI) flanked by 5 'and 3' homology arms.
An msDNA expression vector was generated that had the same HDR-GOI-HDR sequence as used in the conventional plasmid, and two super sequence sites flanking this sequence. msDNA (msDNA HDR-GOI-HDR) containing the HDR-GOI-HDR was then produced in an inducible escherichia coli vector production system using the methods described herein and in U.S. patent nos. 9,290,778 and 9,862,954.
Induced pluripotent stem cells (ipscs) were transfected with equimolar concentrations of plasmid DNA HDR-GOI-HDR or msDNA HDR-GOI-HDR and CRISPR gene editing systems to mediate homologous directed repair knock-in (HDR KI) of GOI.
On days 3, 7 and 15 post-transfection, the total number of intact healthy ipscs expressing GOI on their surface was counted by Fluorescence Activated Cell Sorting (FACS) relative to the total number of transfected cells to assess homologous targeted repair knock-in (HDR KI) efficiency of the GOI. As shown in Q3 of fig. 27B, 28B and 29A, the HDR KI efficiencies of conventional plasmids at 3, 7 and 15 days post-transfection were 8.60%, 7.76% and 8.05%, respectively. As shown in Q3 of fig. 27C, 28C and 29B, higher HDR KI efficiencies were observed for msDNA at 3, 7 and 15 days post-transfection, 15.4% and 15.7%, respectively.
EXAMPLE 7 expression vectors containing modifications of regulatory sequences
A. Expression vector
Expression vectors containing two super sequence sites, a CMV enhancer/promoter, an engineered 5' utr containing an internal minimal intron sequence, and a polygenic expression cassette encoding eGFP and Nluc (as described in examples 1 and 2) were also designed to contain: a 3' UTR (i.e., 2huBGpA-A120, SEQ ID NO: 17) containing two copies of the human β -globin polyadenylation signal and 120 adenine nucleotides, and one or more of the following: (1) A synthetic enhancer 5' to the CMV enhancer (i.e., enhancer-1 (E1)), SEQ ID NO:12 (2) WPRE at the 5 'end of the 3' utr, (3) SRF-UCOE at the 3 'end of the 5' super sequence; and (4) human CSP-B MAR (huMAR) located at the 3' end of eGFP. The pattern of the designed vector is shown in FIGS. 30-38. FIG. 30 shows a map of an expression vector (SS-CMV-UTR 1-SecNLuc-2A-eGFP-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 24) containing the 3' UTR. FIG. 31 shows a map of an expression vector (SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 25) comprising the E1 and 3' UTRs. FIG. 32 shows a map of an expression vector (SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 26) comprising the E1, WPRE and 3' UTR. FIG. 33 shows a map of an expression vector (SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 27) comprising the UCOE, E1, WPRE and 3' UTR. FIG. 34 shows a map of an expression vector (SS. Times. -E1-CMV-UTR1-SecNLuc-2A-eGFP-huMAR-3'UTR [2hBGpA-A120] -SS. Times., SEQ ID NO: 28) comprising the E1, huMAR and 3' UTR. FIG. 35 shows a map of an expression vector (SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-huMAR-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 29) comprising the UCOE, E1, huMAR and 3' UTR. FIG. 36 shows a map of an expression vector (SS-UCOE-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 30) comprising the UCOE, E1, WPRE and 3' UTR. FIG. 37 shows a map of an expression vector (SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-huMAR-WPRE-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 31) comprising the E1, huMAR, WPRE and 3' UTR. FIG. 38 shows a map of an expression vector (SS. Times. -UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-huMAR-WPRE-3'UTR [2hBGpA-A120] -SS, SEQ ID NO: 32) comprising the UCOE, E1, huMAR, WPRE and 3' UTR.
B. Luciferase expression levels
HEK293 cells were transfected with (1) the conventional plasmid pcDNA-CMV-5'UTR-SecNLuc-P2A-eGFP-bGHpA, (2) SS-CMV-UTR 1-SecNLuc-2A-eGFP-3' UTR [2hbgpA-A120] -SS, (3) SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-3'UTR [2hbgpA-A120] -SS, and (4) SS-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3' UTR [2hbgpA-A120] -SS, respectively, by standard procedure.
Luciferase expression was assessed by measuring the intensity of secreted luciferase in the medium in which the cells were cultured on days 2, 3, 7, 10, 14, 21 and 28 post electroporation, as described in example 2B. Referring to fig. 39, the expression levels in the medium of pcDNA-CMV-5' UTR-SecNLuc-P2A-eGFP-bGHpA (conventional pDNA CMV-U), SS x-CMV-UTR 1-SecNLuc-2A-eGFP-3' UTR [2hbgpA-A120] -SS (a: CMV-U1-3' UTR), SS x-E1-CMV-UTR 1-SecNLuc-2A-eGFP-3' UTR [2hbgpA-A120] -SS (B: E1-CMV-U1-3' UTR), and SS x-E1-CMV-UTR 1-SecNLuc-2A-eGFP-WPRE-3' UTR [2hbgpA-A120] -SS (C: E1-U1-WPRE-3 ' UTR) transfected cells are shown.
As shown in FIG. 39, luciferase expression was increased and sustained in the msDNA expression vector containing the 3' UTR as compared to the conventional plasmid having the same promoter and the polygene expression cassette. Expression is further increased by adding E1 (A vs B) and WPRE (B vs C) genetic elements to the msDNA expression vector, and the additive effect of E1 and WPRE genetic elements results in the highest luciferase expression associated with construct C. Statistical analysis was performed using t-test. * =p <0.05 and x=p <0.01.
C. Amplification of the vector to daughter cells and luciferase expression
HEK293 cells were transfected with the four expression vectors described in example B. Cells were passaged once every 7 days for five total passages. At the time of cell passage, the cells were re-seeded at 1/10 of the original cell density. For each cell passage, secreted luciferase expression was measured as described in example 2B. Referring to fig. 40, the expression levels in the medium of cells transfected with msDNA expression vectors are shown compared to conventional plasmids. Statistical analysis and p-values were as described in example B.
At each generation, luciferase expression was detected in cells transfected with any of the msDNA expression vectors, indicating that these vectors were transmitted to daughter cells and had persistent luciferase expression.
As shown in FIG. 40, the additive effect of E1 and WPRE genetic elements resulted in the highest luciferase expression associated with construct C at each generation and the most durable expression after multiple passages.
Example 8 expression vectors containing synthetic promoter sequences
A. Expression vector
Five synthetic promoter sequences were generated: (1) CAG [ E1X3+CBA promoter+intron ] (SEQ ID NO: 35), containing three copies of synthetic enhancer E1 (i.e., 3 copies of SEQ ID NO: 12), chicken beta-actin promoter and chimeric intron, (2) CAG [ E2+CBA promoter+intron ] (SEQ ID NO: 36), containing E2 (U100), chicken beta-actin promoter and chimeric intron, (3) CAG [ E1X3+CBA promoter+UTR1 ] (SEQ ID NO: 37), containing three copies of synthetic enhancer E1, chicken beta-actin promoter and 5' UTR1 (i.e., SEQ ID NO: 2), (4) CAG [ E2 (U100) +CBA promoter+UTR1 ] (SEQ ID NO: 38), containing E2 (U100), chicken beta-actin promoter and 5' UTR1, and (5) CMV enhancer-EF 1-UTR1 (SEQ ID NO: 37), containing short UTR1, and short UTR1 ' UTR1 promoters.
Conventional plasmids containing the CMV enhancer, chicken β -actin promoter, chimeric introns and polygenic expression cassettes encoding eGFP and Nluc were generated as described in examples 1 and 2. A map of the conventional plasmid is shown in FIG. 41 (pGL 2-CAG-SecNLuc-2A-eGFP-WPRE-bGlobal polyA, SEQ ID NO: 34).
msDNA expression vectors containing two super sequence sites, CMV enhancer, chicken β -actin promoter, chimeric intron, multiple gene expression cassettes encoding eGFP and Nluc, WPRE and 3' utr were generated. The vector map is shown in FIG. 42 (4-1 pGL 2-SS-CAG [ CMV enhancer+CBA promoter+intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS, SEQ ID NO: 40).
Five msDNA expression vectors were generated containing two super sequence sites, a polygenic expression cassette encoding eGFP and Nluc, WPRE and 3'UTR, and one of the synthetic promoters (1) - (5) described above, the maps of which were shown in fig. 43 (4-2 pGL 2-SS-CAG [ E1X 3+ CBA promoter + intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120poly a) -SS, SEQ ID NO: 41), fig. 44 (4-3 pGL 2-SS-CAG [ E2 (U100) +cba promoter + intron ] -secgfp-re-3 'UTR (108 to 120 a) -SS, SEQ ID NO: 42), fig. 45 (4-4 l 2-SS-CAG [ E1X 3+ CBA promoter + UTR1] -secgfp-p-re-3' UTR (108 to 120 a) -SS), fig. 44 (4-3 pGL 2A-SS-5 'UTR promoter + CBA) -SS (5) and (4-3 pGL 2A-5) promoter + c-SS-3' UTR (U100) +5) and (4-g 2-SS-5) respectively.
B. Luciferase expression levels
HEK293 cells at 1X10 5 Individual cells/well were seeded in 24-well plates and transfected with 0.25pmol of DNA/well using lipofectamine and the complex of vector described in example a, respectively. Such asSecreted luciferase expression was measured on days 3 and 6 post-transfection as described in example 2B. Referring to fig. 48, the expression levels in the medium of cells transfected with msDNA expression vectors are shown as compared to conventional plasmids. Statistical analysis was performed using a two-way Anova analysis, relative to conventional plasmids. * =p<0.05，**＝p<0.01，****＝p<0.0001。
All msDNA expression vectors have higher luciferase expression levels compared to conventional plasmids. Highest expression was observed with the 4-6-pGL 2-SS-CMV enhancer comprising the EF-1 promoter element and CMV enhancer and 5' UTR 1-EF 1-UTR1-SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120poly a (4-6: CMV-EF1-UTR1-W-3' UTR).
EXAMPLE 9 SS and expression cassette modification
The effect of modification of the Super Sequences (SS) and expression cassettes of the expression vectors of the present disclosure was evaluated based on transfection efficiency, expression of the nucleic acid sequences of interest (including reporter genes, such as the polygenic GFP and luciferase expression cassettes as described in examples 1 and 2), and persistence/amplification of the vector in dividing cells, including fast and slow dividing cells. Furthermore, modifications of SS were evaluated for restriction enzyme activity at SS sites.
Modifications will include individual modifications and combinations of modifications such as, but not limited to: an endonuclease target sequence integrated in the recombinase non-binding region of the SS between the carrier backbone and the recombinase cleavage site; a CAG promoter integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase (i.e., the 3' end of the 5 'ss) and the 5' end of the promoter; a CMV enhancer integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase (i.e., the 3' end of the 5 'ss) and the 5' end of the promoter; enhancer-1 sequences located 5 'to the CMV enhancer and/or 3' to the UCOE; CMV, EF1, SV40, CAG, rho, VDM2, HCR or HLP promoters or variants thereof, CMV promoter variants, EF 1-a promoters, synthetic promoters; integrating a 5'utr comprising an intron located between the promoter and the nucleic acid sequence of interest in the expression cassette, wherein the intron has integrated or has not integrated a non-coding sequence (e.g., a 5' utr comprising a nucleic acid sequence of any of SEQ ID NOs: 2-5); a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal; integrating a woodchuck hepatitis virus post-transcriptional regulatory element located between the nucleic acid of interest and the polyadenylation signal in the expression cassette; a scaffold/matrix attachment region located between the nucleic acid of interest and the polyadenylation signal incorporated into the expression cassette; a ubiquitous chromatin opening element 5' to the promoter in the expression cassette (e.g., 3' to the 5' ss and prior to other sequences in the expression cassette); integration of the 3' UTR located between the nucleic acid of interest and the 3' SS in the expression cassette, for example directly after the stop codon (e.g., a 3' UTR comprising the nucleic acid sequence of any one of SEQ ID NOS: 13-16); and/or a poly (a) tail comprising 100 to 120 adenine nucleotides (e.g., as the 3 'end of the 3' utr).
Sequence(s)
SEQ ID NO. 1 Artificial intron
gtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcag
SEQ ID NO:2 5'UTR1
ctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttcca
SEQ ID NO 3' UTR1 with MAR-5 insertion
ctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtatccatagctgattggtctaaaatgagatacatcaacgctcctccatgttttttgttttctttttaaatgaaaaactttattttttaagaggagtttcaggttcatagcaaaattgagaggaaggtacattcaagctgaggaagttttcctctattcctagtttactgagagattgcatcatgaatgggtgttaaattttgtcaaatgctttttctgtgtctatcaatatgaccatgtgattttcttctttaacctgttgatgggacaaattacgttaattgattttcaaacgttgaaccacccttacatatctggaataaattctacttggttgtggtgtatattttttgatacattcttggattctttttgctaatattttgttgaaaatgtttgtatctttgttcatgagagatattggtctgttgttttcttttcttgtaatgtcattttctagttccggtattaaggtaatgctggcctagttgaatgatttaggaagtattccctctgcttctgtcttctgaaagagattgtagaaagttgatacaatttttttttctttaaatatcttgatagtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttcca
SEQ ID NO:4 5'UTR
attgggatcttcacacagcaggtaaggttgcgggccgggcctgggccgggtccgggccgggccgcactgacccctggtgttgctttttttttttaggccgcaagctgaagcgtgtcc
SEQ ID NO 5'UTR2 (5' UTR of SEQ ID NO 4 with MAR-5 insert)
attgggatcttcacacagcaggtaaggttgcgggccgggcctgggccgggtccgggccgggtatccatagctgattggtctaaaatgagatacatcaacgctcctccatgttttttgttttctttttaaatgaaaaactttattttttaagaggagtttcaggttcatagcaaaattgagaggaaggtacattcaagctgaggaagttttcctctattcctagtttactgagagattgcatcatgaatgggtgttaaattttgtcaaatgctttttctgtgtctatcaatatgaccatgtgattttcttctttaacctgttgatgggacaaattacgttaattgattttcaaacgttgaaccacccttacatatctggaataaattctacttggttgtggtgtatattttttgatacattcttggattctttttgctaatattttgttgaaaatgtttgtatctttgttcatgagagatattggtctgttgttttcttttcttgtaatgtcattttctagttccggtattaaggtaatgctggcctagttgaatgatttaggaagtattccctctgcttctgtcttctgaaagagattgtagaaagttgatacaatttttttttctttaaatatcttgatagccgcactgacccctggtgttgctttttttttttaggccgcaagctgaagcgtgtcc
SEQ ID NO. 6A2UCOE element
gcggccgcacgcgtggccctccgcgcctacagctcaagccacatccgaagggggagggagccgggagctgcgcgcggggccgccggggggaggggtggcaccgcccacgccgggcggccacgaagggcggggcagcgggcgcgcgcgcggcggggggaggggccggcgccgcgcccgctgggaattggggccctagggggagggcggaggcgccgacgaccgcggcacttaccgttcgcggcgtggcgcccggtggtccccaaggggagggaagggggaggcggggcgaggacagtgaccggagtctcctcagcggtggcttttctgcttggcagcctcagcggctggcgccaaaaccggactccgcccacttcctcgcccgccggtgcgagggtgtggaatcctccagacgctgggggagggggagttgggagcttaaaaactagtacccctttgggaccactttcagcagcgaactctcctgtacaccaggggtcagttccacagacgcgggccaggggtgggtcattgcggcgtgaacaataatttgactagaagttgattcgggtgtttccggaaggggccgagtcaatccgccgagttggggcacggaaaacaaaaagggaaggctactaagatttttctggcgggggttatcattggcgtaactgcagggaccacctcccgggttgagggggctggatctccaggctgcggattaagcccctcccgtcggcgttaatttcaaactgcgcgacgtttctcacctgccttcgccaaggcaggggccgggaccctattccaagaggtagtaactagcaggactctagccttccgcaattcattgagcgcatttacggaagtaacgtcgggtactgtctctggccgcaagggtgggaggagtacgcatttggcgtaaggtggggcgtagagccttcccgccattggcggcggatagggcgtttacgcgacggcctgacgtagcggaagacgccttagtgggggggaaggttctagaaaagcggcggcagcggctctagcggcagtagcagcagcgccgggtcccgtgcggaggtgctcctcgcagagttgtttctccagcagcggcagttctcactacagcgccaggacgagtccggttcgtgttcgtccgcggagatctctctcatctcgctcggctgcgggaaatcgggctgaagcgactgagtccgcgatggaggtaacgggtttgaaatcaatgagttattgaaaagggcatggcgaggccgttggcgcctcagtggaagtcggccagccgcctccgtgggagagaggcaggaaatcggaccaattcagtagcagtggggcttaaggtttatgaacggggtcttgagcggaggcctgagcgtacaaacagcttccccaccctcagcctcccggcgccatttcccttcactgggggtgggggatggggagctttcacatggcggacgctgccccgctggggtgaaagtggggcgcggaggcgggacttcttattccctttctaaagcacgctgcttcgggggccacggcgtctcctcggacggccgggcgcgcc
SEQ ID NO:7SRF-UCOEgcacacgaccacaattccactgaaagcattttaatacggaacttgtcactcccagggagcctccgctcagccggcagttggttcatttcaatccccacgacaacccttcaaagtgcagggcagacagcaggtggctctgcccaggcgcctggatcacagcccggcctgcagccctcacctgggcgcggggagaccctgaggacgctcctccaggcggcgctggccggggcctgcggacacggacgggcgggctgagctccgggacccctccccgcgccccgcaccccgcaccccgcaccccgcaccccgcacccggcgctcacccgtcccagccccgccgcccgcagccccagctgcaacgcagccaccgccgccatcgcacccggccccgcgggcgcttccgggacgcaggaggcatctgcatccggggcgccgctgagtcccgcccagagccccgcccccggctccaggttctgcgagcggcttccgccgggctgctccgcgggcgcgtcggccatgagcgagttgccgggcgacgtgcgggcgtttctgcgggagcacccgagcctgcggctccagacggacgcccgcaaggttcgcagcgcgggaggggaacggagtggcggagaagggcgcagttgggatgaggggctgaggggagggcaggggagaggagagggcaggggagaggggagaggggagagcaggagagaggggaaggcaggggagagggcgcggcgggatcaggggaggagagggaa
SEQ ID NO. 8cHS4 insulator
ggggagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgaccgcccggggctccgctccggtccggcgctccccccgcatcccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctgggggatacggggaaaaggggagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgaccgcccggggctccgctccggtccggcgctccccccgcatcccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctgggggatacggggaaaa
SEQ ID NO:9MAR-5
tatccatagctgattggtctaaaatgagatacatcaacgctcctccatgttttttgttttctttttaaatgaaaaactttattttttaagaggagtttcaggttcatagcaaaattgagaggaaggtacattcaagctgaggaagttttcctctattcctagtttactgagagattgcatcatgaatgggtgttaaattttgtcaaatgctttttctgtgtctatcaatatgaccatgtgattttcttctttaacctgttgatgggacaaattacgttaattgattttcaaacgttgaaccacccttacatatctggaataaattctacttggttgtggtgtatattttttgatacattcttggattctttttgctaatattttgttgaaaatgtttgtatctttgttcatgagagatattggtctgttgttttcttttcttgtaatgtcattttctagttccggtattaaggtaatgctggcctagttgaatgatttaggaagtattccctctgcttctgtcttctgaaagagattgtagaaagttgatacaatttttttttctttaaatatcttgatag
SEQ ID NO 10 human CSP-B MAR (huMAR)
ggatcccattctccttgatgtactaatttttctttaaaagtgataataatagctcccatttagaatttttaaataacacaacaaatgtaaagtaactaatgtgtcctctggatcatggtaagtaatgaataaatttaactccctttaccttctccctttgctattttttccatgctaggatttatacatttttaaaaaactaaatctgctatcaaatgacagctttaaatttactttttaaaatttgttattgtatatatttatggggtataaagtgatgttatgatatatatatacacaatgtacactgattaaatcaagccaattaacattttatcatctcaaatacttaacattttttgtagtgagaacatttgaaatttacttttagcaatttcaaaacatacattattattattaactatagtcaccatgatgtaccatagatctttaaaaacttattcttcctgcctaactgaaactttgtactctttgactaacatcttttcattcccccacttcccagcctctggtaatcaccattacacactctgcttctatgagttcaattgctttagactccacgtaataaatgagatcatgcagcatttggctttctgtgcctggcttatccttgcttagcatggtgtcttacaggttcatccatgttgcaacaaataacagaatctcattctttgttaaggctgaatactattccattgggtatatataccacattttccttatccattaatccactgatggacccttaggttgttgattccatatattggctattgtaaatagtgcagcaatgaacatgagagtgcaactatctcttcaatgtactgatttcgaatccttcggatctatctcagaagtgagattgcaggatcatataattctacttttagtcttttgaggagctccatacagctttccatatggccatactaattacattctcatcaacagtgtacaatggtttccttttctccacatcctcaccaacatttataattttttgtctttttgataatagccatctgacaggtgtaaagtgatagctcattgcagttttaatttgcattttttgatgattagtaatgttgagaattttttcatatatctcttggccagttgcatgtcttctttggaaaaatgtctattcagttcctttgcccattttttaattgggatttttggtttcttgctattgagttgtttgaattc
SEQ ID NO:11WPRE
Tcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctg
SEQ ID NO. 12 enhancer-1
gggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcac
13 copies of Xenopus laevis beta-globin polyadenylation signal (2 xlBGpA) of SEQ ID NO. 13
aaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacaaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcac
14 2 copies of human beta-globin polyadenylation signal (2 huBGpA) SEQ ID NO
gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaa
Xenopus laevis and human beta-globin polyadenylation signal (xlhuBGpA) heterozygous for SEQ ID NO 15
aaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaa
SEQ ID NO:16 2xlBGpA-A120
aaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacaaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
SEQ ID NO:17 2huBGpA-A120
gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
SEQ ID NO:18xlhuBGpA-A120
aaccagcctcaagaacacccgaatggagtctctaagctacataataccaacttacactttacaaaatgttgtcccccaaaatgtagccattcgtatctgctcctaataaaaagaaagtttcttcacgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
SEQ ID NO:19pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*
ccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagcggccgcactcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaattggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:20 pcDNA-CMV-5'UTR-SecNLuc-P2A-eGFP-bGHpA
gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccctgccttctccctcctgtgagtttggtaagtcactgactgtctatgcctgggaaagggtgggcaggagatggggcagtgcaggaaaagtggcactatgaaccctgcagccctaggaatgcatctagacaattgtactaaccttcttctctttcctctcctgacaggttggtgtacagtagcttccactcctgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggaattctgcagatatccagcacagtggcggccgctcgagtctagaggaagcggagctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctatgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgtacaagtaagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc
SEQ ID NO:21 pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS*taaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaattggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacca
SEQ ID NO:22 pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*
taaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaattggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacca
SEQ ID NO:23 pGL2-SS*-CMV-UTR2-SecNLuc-2A-eGFP-WPRE-BGpA-SS*
aattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctattgggatcttcacacagcaggtaaggttgcgggccgggcctgggccgggtccgggccgggtatccatagctgattggtctaaaatgagatacatcaacgctcctccatgttttttgttttctttttaaatgaaaaactttattttttaagaggagtttcaggttcatagcaaaattgagaggaaggtacattcaagctgaggaagttttcctctattcctagtttactgagagattgcatcatgaatgggtgttaaattttgtcaaatgctttttctgtgtctatcaatatgaccatgtgattttcttctttaacctgttgatgggacaaattacgttaattgattttcaaacgttgaaccacccttacatatctggaataaattctacttggttgtggtgtatattttttgatacattcttggattctttttgctaatattttgttgaaaatgtttgtatctttgttcatgagagatattggtctgttgttttcttttcttgtaatgtcattttctagttccggtattaaggtaatgctggcctagttgaatgatttaggaagtattccctctgcttctgtcttctgaaagagattgtagaaagttgatacaatttttttttctttaaatatcttgatagccgcactgacccctggtgttgctttttttttttaggccgcaagctgaagcgtgtccgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaattggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacca
SEQ ID NO:24 SS*-CMV-UTR1-SecNLuc-2A-eGFP-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:25 SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:26 SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:27 SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgcacacgaccacaattccactgaaagcattttaatacggaacttgtcactcccagggagcctccgctcagccggcagttggttcatttcaatccccacgacaacccttcaaagtgcagggcagacagcaggtggctctgcccaggcgcctggatcacagcccggcctgcagccctcacctgggcgcggggagaccctgaggacgctcctccaggcggcgctggccggggcctgcggacacggacgggcgggctgagctccgggacccctccccgcgccccgcaccccgcaccccgcaccccgcaccccgcacccggcgctcacccgtcccagccccgccgcccgcagccccagctgcaacgcagccaccgccgccatcgcacccggccccgcgggcgcttccgggacgcaggaggcatctgcatccggggcgccgctgagtcccgcccagagccccgcccccggctccaggttctgcgagcggcttccgccgggctgctccgcgggcgcgtcggccatgagcgagttgccgggcgacgtgcgggcgtttctgcgggagcacccgagcctgcggctccagacggacgcccgcaaggttcgcagcgcgggaggggaacggagtggcggagaagggcgcagttgggatgaggggctgaggggagggcaggggagaggagagggcaggggagaggggagaggggagagcaggagagaggggaaggcaggggagagggcgcggcgggatcaggggaggagagggaagggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:28 SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-huMAR-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggatcccattctccttgatgtactaatttttctttaaaagtgataataatagctcccatttagaatttttaaataacacaacaaatgtaaagtaactaatgtgtcctctggatcatggtaagtaatgaataaatttaactccctttaccttctccctttgctattttttccatgctaggatttatacatttttaaaaaactaaatctgctatcaaatgacagctttaaatttactttttaaaatttgttattgtatatatttatggggtataaagtgatgttatgatatatatatacacaatgtacactgattaaatcaagccaattaacattttatcatctcaaatacttaacattttttgtagtgagaacatttgaaatttacttttagcaatttcaaaacatacattattattattaactatagtcaccatgatgtaccatagatctttaaaaacttattcttcctgcctaactgaaactttgtactctttgactaacatcttttcattcccccacttcccagcctctggtaatcaccattacacactctgcttctatgagttcaattgctttagactccacgtaataaatgagatcatgcagcatttggctttctgtgcctggcttatccttgcttagcatggtgtcttacaggttcatccatgttgcaacaaataacagaatctcattctttgttaaggctgaatactattccattgggtatatataccacattttccttatccattaatccactgatggacccttaggttgttgattccatatattggctattgtaaatagtgcagcaatgaacatgagagtgcaactatctcttcaatgtactgatttcgaatccttcggatctatctcagaagtgagattgcaggatcatataattctacttttagtcttttgaggagctccatacagctttccatatggccatactaattacattctcatcaacagtgtacaatggtttccttttctccacatcctcaccaacatttataattttttgtctttttgataatagccatctgacaggtgtaaagtgatagctcattgcagttttaatttgcattttttgatgattagtaatgttgagaattttttcatatatctcttggccagttgcatgtcttctttggaaaaatgtctattcagttcctttgcccattttttaattgggatttttggtttcttgctattgagttgtttgaattcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:29 SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-huMAR-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgcacacgaccacaattccactgaaagcattttaatacggaacttgtcactcccagggagcctccgctcagccggcagttggttcatttcaatccccacgacaacccttcaaagtgcagggcagacagcaggtggctctgcccaggcgcctggatcacagcccggcctgcagccctcacctgggcgcggggagaccctgaggacgctcctccaggcggcgctggccggggcctgcggacacggacgggcgggctgagctccgggacccctccccgcgccccgcaccccgcaccccgcaccccgcaccccgcacccggcgctcacccgtcccagccccgccgcccgcagccccagctgcaacgcagccaccgccgccatcgcacccggccccgcgggcgcttccgggacgcaggaggcatctgcatccggggcgccgctgagtcccgcccagagccccgcccccggctccaggttctgcgagcggcttccgccgggctgctccgcgggcgcgtcggccatgagcgagttgccgggcgacgtgcgggcgtttctgcgggagcacccgagcctgcggctccagacggacgcccgcaaggttcgcagcgcgggaggggaacggagtggcggagaagggcgcagttgggatgaggggctgaggggagggcaggggagaggagagggcaggggagaggggagaggggagagcaggagagaggggaaggcaggggagagggcgcggcgggatcaggggaggagagggaagggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggatcccattctccttgatgtactaatttttctttaaaagtgataataatagctcccatttagaatttttaaataacacaacaaatgtaaagtaactaatgtgtcctctggatcatggtaagtaatgaataaatttaactccctttaccttctccctttgctattttttccatgctaggatttatacatttttaaaaaactaaatctgctatcaaatgacagctttaaatttactttttaaaatttgttattgtatatatttatggggtataaagtgatgttatgatatatatatacacaatgtacactgattaaatcaagccaattaacattttatcatctcaaatacttaacattttttgtagtgagaacatttgaaatttacttttagcaatttcaaaacatacattattattattaactatagtcaccatgatgtaccatagatctttaaaaacttattcttcctgcctaactgaaactttgtactctttgactaacatcttttcattcccccacttcccagcctctggtaatcaccattacacactctgcttctatgagttcaattgctttagactccacgtaataaatgagatcatgcagcatttggctttctgtgcctggcttatccttgcttagcatggtgtcttacaggttcatccatgttgcaacaaataacagaatctcattctttgttaaggctgaatactattccattgggtatatataccacattttccttatccattaatccactgatggacccttaggttgttgattccatatattggctattgtaaatagtgcagcaatgaacatgagagtgcaactatctcttcaatgtactgatttcgaatccttcggatctatctcagaagtgagattgcaggatcatataattctacttttagtcttttgaggagctccatacagctttccatatggccatactaattacattctcatcaacagtgtacaatggtttccttttctccacatcctcaccaacatttataattttttgtctttttgataatagccatctgacaggtgtaaagtgatagctcattgcagttttaatttgcattttttgatgattagtaatgttgagaattttttcatatatctcttggccagttgcatgtcttctttggaaaaatgtctattcagttcctttgcccattttttaattgggatttttggtttcttgctattgagttgtttgaattcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:30 SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgcacacgaccacaattccactgaaagcattttaatacggaacttgtcactcccagggagcctccgctcagccggcagttggttcatttcaatccccacgacaacccttcaaagtgcagggcagacagcaggtggctctgcccaggcgcctggatcacagcccggcctgcagccctcacctgggcgcggggagaccctgaggacgctcctccaggcggcgctggccggggcctgcggacacggacgggcgggctgagctccgggacccctccccgcgccccgcaccccgcaccccgcaccccgcaccccgcacccggcgctcacccgtcccagccccgccgcccgcagccccagctgcaacgcagccaccgccgccatcgcacccggccccgcgggcgcttccgggacgcaggaggcatctgcatccggggcgccgctgagtcccgcccagagccccgcccccggctccaggttctgcgagcggcttccgccgggctgctccgcgggcgcgtcggccatgagcgagttgccgggcgacgtgcgggcgtttctgcgggagcacccgagcctgcggctccagacggacgcccgcaaggttcgcagcgcgggaggggaacggagtggcggagaagggcgcagttgggatgaggggctgaggggagggcaggggagaggagagggcaggggagaggggagaggggagagcaggagagaggggaaggcaggggagagggcgcggcgggatcaggggaggagagggaagggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:31 SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-MAR-WPRE-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggatcccattctccttgatgtactaatttttctttaaaagtgataataatagctcccatttagaatttttaaataacacaacaaatgtaaagtaactaatgtgtcctctggatcatggtaagtaatgaataaatttaactccctttaccttctccctttgctattttttccatgctaggatttatacatttttaaaaaactaaatctgctatcaaatgacagctttaaatttactttttaaaatttgttattgtatatatttatggggtataaagtgatgttatgatatatatatacacaatgtacactgattaaatcaagccaattaacattttatcatctcaaatacttaacattttttgtagtgagaacatttgaaatttacttttagcaatttcaaaacatacattattattattaactatagtcaccatgatgtaccatagatctttaaaaacttattcttcctgcctaactgaaactttgtactctttgactaacatcttttcattcccccacttcccagcctctggtaatcaccattacacactctgcttctatgagttcaattgctttagactccacgtaataaatgagatcatgcagcatttggctttctgtgcctggcttatccttgcttagcatggtgtcttacaggttcatccatgttgcaacaaataacagaatctcattctttgttaaggctgaatactattccattgggtatatataccacattttccttatccattaatccactgatggacccttaggttgttgattccatatattggctattgtaaatagtgcagcaatgaacatgagagtgcaactatctcttcaatgtactgatttcgaatccttcggatctatctcagaagtgagattgcaggatcatataattctacttttagtcttttgaggagctccatacagctttccatatggccatactaattacattctcatcaacagtgtacaatggtttccttttctccacatcctcaccaacatttataattttttgtctttttgataatagccatctgacaggtgtaaagtgatagctcattgcagttttaatttgcattttttgatgattagtaatgttgagaattttttcatatatctcttggccagttgcatgtcttctttggaaaaatgtctattcagttcctttgcccattttttaattgggatttttggtttcttgctattgagttgtttgaattcaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:32 SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-MAR-WPRE-3'UTR[2huBGpA-A120]-SS*
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgcacacgaccacaattccactgaaagcattttaatacggaacttgtcactcccagggagcctccgctcagccggcagttggttcatttcaatccccacgacaacccttcaaagtgcagggcagacagcaggtggctctgcccaggcgcctggatcacagcccggcctgcagccctcacctgggcgcggggagaccctgaggacgctcctccaggcggcgctggccggggcctgcggacacggacgggcgggctgagctccgggacccctccccgcgccccgcaccccgcaccccgcaccccgcaccccgcacccggcgctcacccgtcccagccccgccgcccgcagccccagctgcaacgcagccaccgccgccatcgcacccggccccgcgggcgcttccgggacgcaggaggcatctgcatccggggcgccgctgagtcccgcccagagccccgcccccggctccaggttctgcgagcggcttccgccgggctgctccgcgggcgcgtcggccatgagcgagttgccgggcgacgtgcgggcgtttctgcgggagcacccgagcctgcggctccagacggacgcccgcaaggttcgcagcgcgggaggggaacggagtggcggagaagggcgcagttgggatgaggggctgaggggagggcaggggagaggagagggcaggggagaggggagaggggagagcaggagagaggggaaggcaggggagagggcgcggcgggatcaggggaggagagggaagggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggatcccattctccttgatgtactaatttttctttaaaagtgataataatagctcccatttagaatttttaaataacacaacaaatgtaaagtaactaatgtgtcctctggatcatggtaagtaatgaataaatttaactccctttaccttctccctttgctattttttccatgctaggatttatacatttttaaaaaactaaatctgctatcaaatgacagctttaaatttactttttaaaatttgttattgtatatatttatggggtataaagtgatgttatgatatatatatacacaatgtacactgattaaatcaagccaattaacattttatcatctcaaatacttaacattttttgtagtgagaacatttgaaatttacttttagcaatttcaaaacatacattattattattaactatagtcaccatgatgtaccatagatctttaaaaacttattcttcctgcctaactgaaactttgtactctttgactaacatcttttcattcccccacttcccagcctctggtaatcaccattacacactctgcttctatgagttcaattgctttagactccacgtaataaatgagatcatgcagcatttggctttctgtgcctggcttatccttgcttagcatggtgtcttacaggttcatccatgttgcaacaaataacagaatctcattctttgttaaggctgaatactattccattgggtatatataccacattttccttatccattaatccactgatggacccttaggttgttgattccatatattggctattgtaaatagtgcagcaatgaacatgagagtgcaactatctcttcaatgtactgatttcgaatccttcggatctatctcagaagtgagattgcaggatcatataattctacttttagtcttttgaggagctccatacagctttccatatggccatactaattacattctcatcaacagtgtacaatggtttccttttctccacatcctcaccaacatttataattttttgtctttttgataatagccatctgacaggtgtaaagtgatagctcattgcagttttaatttgcattttttgatgattagtaatgttgagaattttttcatatatctcttggccagttgcatgtcttctttggaaaaatgtctattcagttcctttgcccattttttaattgggatttttggtttcttgctattgagttgtttgaattcaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO. 33 (super sequence, SS)
taaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacc
SEQ ID NO:34pGL2-CAG-SecNLuc-2A-eGFP-WPRE-bGlobin polyA
cccgggaggtaccgagctcttacgcgtgctagcctgggtcgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaagcggccgcactcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggacattggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO:35CAG [ E1X3+CBA promoter+intron ]
gggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacag
36CAG [ E2+CBA promoter+intron ]
tgggactttccactagacatgacacagcaatctgatatgcttgcgtgagaagaggattcatatcctgggactttccacagattttaccggaagttgttagatgcttgcgtgagaagatctaacatgacacagcaatccttagtgggactttccaagtatgtggggcggggagtatacatgacacagcaattgatcattaccggaagtttataggtgggactttccagacctatgcttgcgtgagaagaaaggtctgggactttccagtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacag
SEQ ID NO. 37CAG [ E1X3+CBA promoter+UTR1 ]
gggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtc
38CAG [ E2 (U100) +CBA promoter+UTR1 ]
Tgggactttccactagacatgacacagcaatctgatatgcttgcgtgagaagaggattcatatcctgggactttccacagattttaccggaagttgttagatgcttgcgtgagaagatctaacatgacacagcaatccttagtgggactttccaagtatgtggggcggggagtatacatgacacagcaattgatcattaccggaagtttataggtgggactttccagacctatgcttgcgtgagaagaaaggtctgggactttccagtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtc
SEQ ID NO 39CMV enhancer-EF 1-UTR1
Gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtc
SEQ ID NO. 40.4-1 pGL 2-SS-CAG [ CMV enhancer+CBA promoter+intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttc
agcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO. 41 4-2pGL 2-SS-CAG [ E1X3+ CBA promoter+intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO. 42 4-3pGL 2-SS-CAG [ E2 (U100) +CBA promoter+intron ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS ]
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgactgggactttccactagacatgacacagcaatctgatatgcttgcgtgagaagaggattcatatcctgggactttccacagattttaccggaagttgttagatgcttgcgtgagaagatctaacatgacacagcaatccttagtgggactttccaagtatgtggggcggggagtatacatgacacagcaattgatcattaccggaagtttataggtgggactttccagacctatgcttgcgtgagaagaaaggtctgggactttccagtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO. 43 4-4pGL 2-SS-CAG [ E1X3+ CBA promoter +UTR1] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO 44 4-5-pGL 2-SS-CAG [ E2 (U100) +CBA promoter +UTR1] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS ]
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgactgggactttccactagacatgacacagcaatctgatatgcttgcgtgagaagaggattcatatcctgggactttccacagattttaccggaagttgttagatgcttgcgtgagaagatctaacatgacacagcaatccttagtgggactttccaagtatgtggggcggggagtatacatgacacagcaattgatcattaccggaagtttataggtgggactttccagacctatgcttgcgtgagaagaaaggtctgggactttccagtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggaaaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
SEQ ID NO. 45 4-6-pGL 2-SS-CMV enhancer-EF 1-UTR1-SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS
cccgggaggtaccgagctcttacgcgtgctagaattaaagtaacccaatcagcacacaattgccattatacgcgcgtataatggactattgtgtgctgataaacctatttcagcatactacgcgcgtagtatgctgaaataggtgactagaagttcctatactttctagagaataggaacttcataacttcgtataatgtatgctatacgaagttatgggttactttaatttggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacccctgggtcgacgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgccttctccctcctgtgagtttggtaagtcgacgggccgggcctgggccgggtccgggccgggtcgttggatccccactacagcccgatactcaagcttgacgaattcgagtatccaaggtagtggactagtgtgacgctgctgacccctttctttcccttctgcaggttggtgtacagtagcttccaaattgattaattcgagcgaacgcgtcgccaccatgaactccttctccacaagcgccttcggtccagttgccttctccctgggcctgctcctggtgttgcctgctgccttccctgccccagtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggctagcgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggaagcggagagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacctggatccggaatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcgctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaagctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaactcggaaggacatggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaaattaaagtaacccataacttcgtatagcatacattatacgaagttatgaagttcctattctctagaaagtataggaacttctagtcacctatttcagcatactacgcgcgtagtatgctgaaataggtttatcagcacacaatagtccattatacgcgcgtataatggcaattgtgtgctgattgggttactttaatttggatccgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctaccatgataagtaagtaatattaaggtacgtggaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatggtactgtaactgagctaacataa
Three copies of enhancer-1 of SEQ ID NO. 46
gggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcacgggactttccggggcggggcacgtggtgcacgggactttccgtgcacgtgcacgggactttccgggactttccgggactttccgtgcaccacgtggggactttccgtgcac
SEQ ID NO. 47 chimeric intron
ggagtcgctgcgttgccttcgccccgtgccccgctccgcgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggctcgtttcttttctgtggctgcgtgaaagccttaaagggctccgggagggccctttgtgcgggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcggcggtcgggctgtaacccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacag
The scope of the present disclosure is not limited to the specific aspects described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
Other aspects are within the scope of the following claims.
Sequence listing
<110> medical phage biopharmaceutical company (MEDIPHAGE BIOCEUTICALS, INC.)
<120> expression vectors, bacterial sequence-free vectors, and methods of making and using the same
<130> 4471.007PC03
<150> US 63/331,638
<151> 2022-04-15
<150> US 63/306,015
<151> 2022-02-02
<150> US 63/211,343
<151> 2021-06-16
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 144
<212> DNA
<213> artificial sequence
<220>
<223> Artificial intron
<400> 1
gtaagtcgac gggccgggcc tgggccgggt ccgggccggg tcgttggatc cccactacag 60
cccgatactc aagcttgacg aattcgagta tccaaggtag tggactagtg tgacgctgct 120
gacccctttc tttcccttct gcag 144
<210> 2
<211> 190
<212> DNA
<213> artificial sequence
<220>
<223> 5'UTR1
<400> 2
ctgccttctc cctcctgtga gtttggtaag tcgacgggcc gggcctgggc cgggtccggg 60
ccgggtcgtt ggatccccac tacagcccga tactcaagct tgacgaattc gagtatccaa 120
ggtagtggac tagtgtgacg ctgctgaccc ctttctttcc cttctgcagg ttggtgtaca 180
gtagcttcca 190
<210> 3
<211> 771
<212> DNA
<213> artificial sequence
<220>
<223> 5' UTR1 with MAR-5 insertion
<400> 3
ctgccttctc cctcctgtga gtttggtaag tcgacgggcc gggcctgggc cgggtccggg 60
ccgggtatcc atagctgatt ggtctaaaat gagatacatc aacgctcctc catgtttttt 120
gttttctttt taaatgaaaa actttatttt ttaagaggag tttcaggttc atagcaaaat 180
tgagaggaag gtacattcaa gctgaggaag ttttcctcta ttcctagttt actgagagat 240
tgcatcatga atgggtgtta aattttgtca aatgcttttt ctgtgtctat caatatgacc 300
atgtgatttt cttctttaac ctgttgatgg gacaaattac gttaattgat tttcaaacgt 360
tgaaccaccc ttacatatct ggaataaatt ctacttggtt gtggtgtata ttttttgata 420
cattcttgga ttctttttgc taatattttg ttgaaaatgt ttgtatcttt gttcatgaga 480
gatattggtc tgttgttttc ttttcttgta atgtcatttt ctagttccgg tattaaggta 540
atgctggcct agttgaatga tttaggaagt attccctctg cttctgtctt ctgaaagaga 600
ttgtagaaag ttgatacaat ttttttttct ttaaatatct tgatagtcgt tggatcccca 660
ctacagcccg atactcaagc ttgacgaatt cgagtatcca aggtagtgga ctagtgtgac 720
gctgctgacc cctttctttc ccttctgcag gttggtgtac agtagcttcc a 771
<210> 4
<211> 117
<212> DNA
<213> artificial sequence
<220>
<223> 5'UTR
<400> 4
attgggatct tcacacagca ggtaaggttg cgggccgggc ctgggccggg tccgggccgg 60
gccgcactga cccctggtgt tgcttttttt ttttaggccg caagctgaag cgtgtcc 117
<210> 5
<211> 698
<212> DNA
<213> artificial sequence
<220>
<223> 5'UTR2 (5' UTR of SEQ ID NO:4 has MAR-5 insertion)
<400> 5
attgggatct tcacacagca ggtaaggttg cgggccgggc ctgggccggg tccgggccgg 60
gtatccatag ctgattggtc taaaatgaga tacatcaacg ctcctccatg ttttttgttt 120
tctttttaaa tgaaaaactt tattttttaa gaggagtttc aggttcatag caaaattgag 180
aggaaggtac attcaagctg aggaagtttt cctctattcc tagtttactg agagattgca 240
tcatgaatgg gtgttaaatt ttgtcaaatg ctttttctgt gtctatcaat atgaccatgt 300
gattttcttc tttaacctgt tgatgggaca aattacgtta attgattttc aaacgttgaa 360
ccacccttac atatctggaa taaattctac ttggttgtgg tgtatatttt ttgatacatt 420
cttggattct ttttgctaat attttgttga aaatgtttgt atctttgttc atgagagata 480
ttggtctgtt gttttctttt cttgtaatgt cattttctag ttccggtatt aaggtaatgc 540
tggcctagtt gaatgattta ggaagtattc cctctgcttc tgtcttctga aagagattgt 600
agaaagttga tacaattttt ttttctttaa atatcttgat agccgcactg acccctggtg 660
ttgctttttt tttttaggcc gcaagctgaa gcgtgtcc 698
<210> 6
<211> 1579
<212> DNA
<213> artificial sequence
<220>
<223> A2UCOE element
<400> 6
gcggccgcac gcgtggccct ccgcgcctac agctcaagcc acatccgaag ggggagggag 60
ccgggagctg cgcgcggggc cgccgggggg aggggtggca ccgcccacgc cgggcggcca 120
cgaagggcgg ggcagcgggc gcgcgcgcgg cggggggagg ggccggcgcc gcgcccgctg 180
ggaattgggg ccctaggggg agggcggagg cgccgacgac cgcggcactt accgttcgcg 240
gcgtggcgcc cggtggtccc caaggggagg gaagggggag gcggggcgag gacagtgacc 300
ggagtctcct cagcggtggc ttttctgctt ggcagcctca gcggctggcg ccaaaaccgg 360
actccgccca cttcctcgcc cgccggtgcg agggtgtgga atcctccaga cgctggggga 420
gggggagttg ggagcttaaa aactagtacc cctttgggac cactttcagc agcgaactct 480
cctgtacacc aggggtcagt tccacagacg cgggccaggg gtgggtcatt gcggcgtgaa 540
caataatttg actagaagtt gattcgggtg tttccggaag gggccgagtc aatccgccga 600
gttggggcac ggaaaacaaa aagggaaggc tactaagatt tttctggcgg gggttatcat 660
tggcgtaact gcagggacca cctcccgggt tgagggggct ggatctccag gctgcggatt 720
aagcccctcc cgtcggcgtt aatttcaaac tgcgcgacgt ttctcacctg ccttcgccaa 780
ggcaggggcc gggaccctat tccaagaggt agtaactagc aggactctag ccttccgcaa 840
ttcattgagc gcatttacgg aagtaacgtc gggtactgtc tctggccgca agggtgggag 900
gagtacgcat ttggcgtaag gtggggcgta gagccttccc gccattggcg gcggataggg 960
cgtttacgcg acggcctgac gtagcggaag acgccttagt gggggggaag gttctagaaa 1020
agcggcggca gcggctctag cggcagtagc agcagcgccg ggtcccgtgc ggaggtgctc 1080
ctcgcagagt tgtttctcca gcagcggcag ttctcactac agcgccagga cgagtccggt 1140
tcgtgttcgt ccgcggagat ctctctcatc tcgctcggct gcgggaaatc gggctgaagc 1200
gactgagtcc gcgatggagg taacgggttt gaaatcaatg agttattgaa aagggcatgg 1260
cgaggccgtt ggcgcctcag tggaagtcgg ccagccgcct ccgtgggaga gaggcaggaa 1320
atcggaccaa ttcagtagca gtggggctta aggtttatga acggggtctt gagcggaggc 1380
ctgagcgtac aaacagcttc cccaccctca gcctcccggc gccatttccc ttcactgggg 1440
gtgggggatg gggagctttc acatggcgga cgctgccccg ctggggtgaa agtggggcgc 1500
ggaggcggga cttcttattc cctttctaaa gcacgctgct tcgggggcca cggcgtctcc 1560
tcggacggcc gggcgcgcc 1579
<210> 7
<211> 761
<212> DNA
<213> artificial sequence
<220>
<223> SRF-UCOE
<400> 7
gcacacgacc acaattccac tgaaagcatt ttaatacgga acttgtcact cccagggagc 60
ctccgctcag ccggcagttg gttcatttca atccccacga caacccttca aagtgcaggg 120
cagacagcag gtggctctgc ccaggcgcct ggatcacagc ccggcctgca gccctcacct 180
gggcgcgggg agaccctgag gacgctcctc caggcggcgc tggccggggc ctgcggacac 240
ggacgggcgg gctgagctcc gggacccctc cccgcgcccc gcaccccgca ccccgcaccc 300
cgcaccccgc acccggcgct cacccgtccc agccccgccg cccgcagccc cagctgcaac 360
gcagccaccg ccgccatcgc acccggcccc gcgggcgctt ccgggacgca ggaggcatct 420
gcatccgggg cgccgctgag tcccgcccag agccccgccc ccggctccag gttctgcgag 480
cggcttccgc cgggctgctc cgcgggcgcg tcggccatga gcgagttgcc gggcgacgtg 540
cgggcgtttc tgcgggagca cccgagcctg cggctccaga cggacgcccg caaggttcgc 600
agcgcgggag gggaacggag tggcggagaa gggcgcagtt gggatgaggg gctgagggga 660
gggcagggga gaggagaggg caggggagag gggagagggg agagcaggag agaggggaag 720
gcaggggaga gggcgcggcg ggatcagggg aggagaggga a 761
<210> 8
<211> 478
<212> DNA
<213> artificial sequence
<220>
<223> cHS4 insulator
<400> 8
ggggagctca cggggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc 60
ccgctagggg gcagcagcga ccgcccgggg ctccgctccg gtccggcgct ccccccgcat 120
cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt 180
cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctgggggata cggggaaaag 240
gggagctcac ggggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc 300
cgctaggggg cagcagcgac cgcccggggc tccgctccgg tccggcgctc cccccgcatc 360
ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg gatcgctttc 420
ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tgggggatac ggggaaaa 478
<210> 9
<211> 581
<212> DNA
<213> artificial sequence
<220>
<223> MAR-5
<400> 9
tatccatagc tgattggtct aaaatgagat acatcaacgc tcctccatgt tttttgtttt 60
ctttttaaat gaaaaacttt attttttaag aggagtttca ggttcatagc aaaattgaga 120
ggaaggtaca ttcaagctga ggaagttttc ctctattcct agtttactga gagattgcat 180
catgaatggg tgttaaattt tgtcaaatgc tttttctgtg tctatcaata tgaccatgtg 240
attttcttct ttaacctgtt gatgggacaa attacgttaa ttgattttca aacgttgaac 300
cacccttaca tatctggaat aaattctact tggttgtggt gtatattttt tgatacattc 360
ttggattctt tttgctaata ttttgttgaa aatgtttgta tctttgttca tgagagatat 420
tggtctgttg ttttcttttc ttgtaatgtc attttctagt tccggtatta aggtaatgct 480
ggcctagttg aatgatttag gaagtattcc ctctgcttct gtcttctgaa agagattgta 540
gaaagttgat acaatttttt tttctttaaa tatcttgata g 581
<210> 10
<211> 1233
<212> DNA
<213> artificial sequence
<220>
<223> human CSP-B MAR (huMAR)
<400> 10
ggatcccatt ctccttgatg tactaatttt tctttaaaag tgataataat agctcccatt 60
tagaattttt aaataacaca acaaatgtaa agtaactaat gtgtcctctg gatcatggta 120
agtaatgaat aaatttaact ccctttacct tctccctttg ctattttttc catgctagga 180
tttatacatt tttaaaaaac taaatctgct atcaaatgac agctttaaat ttacttttta 240
aaatttgtta ttgtatatat ttatggggta taaagtgatg ttatgatata tatatacaca 300
atgtacactg attaaatcaa gccaattaac attttatcat ctcaaatact taacattttt 360
tgtagtgaga acatttgaaa tttactttta gcaatttcaa aacatacatt attattatta 420
actatagtca ccatgatgta ccatagatct ttaaaaactt attcttcctg cctaactgaa 480
actttgtact ctttgactaa catcttttca ttcccccact tcccagcctc tggtaatcac 540
cattacacac tctgcttcta tgagttcaat tgctttagac tccacgtaat aaatgagatc 600
atgcagcatt tggctttctg tgcctggctt atccttgctt agcatggtgt cttacaggtt 660
catccatgtt gcaacaaata acagaatctc attctttgtt aaggctgaat actattccat 720
tgggtatata taccacattt tccttatcca ttaatccact gatggaccct taggttgttg 780
attccatata ttggctattg taaatagtgc agcaatgaac atgagagtgc aactatctct 840
tcaatgtact gatttcgaat ccttcggatc tatctcagaa gtgagattgc aggatcatat 900
aattctactt ttagtctttt gaggagctcc atacagcttt ccatatggcc atactaatta 960
cattctcatc aacagtgtac aatggtttcc ttttctccac atcctcacca acatttataa 1020
ttttttgtct ttttgataat agccatctga caggtgtaaa gtgatagctc attgcagttt 1080
taatttgcat tttttgatga ttagtaatgt tgagaatttt ttcatatatc tcttggccag 1140
ttgcatgtct tctttggaaa aatgtctatt cagttccttt gcccattttt taattgggat 1200
ttttggtttc ttgctattga gttgtttgaa ttc 1233
<210> 11
<211> 597
<212> DNA
<213> artificial sequence
<220>
<223> WPRE
<400> 11
tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 60
ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 120
cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg 180
agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 240
ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 300
tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 360
ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc 420
tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 480
ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 540
gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctg 597
<210> 12
<211> 111
<212> DNA
<213> artificial sequence
<220>
<223> enhancer-1
<400> 12
gggactttcc ggggcggggc acgtggtgca cgggactttc cgtgcacgtg cacgggactt 60
tccgggactt tccgggactt tccgtgcacc acgtggggac tttccgtgca c 111
<210> 13
<211> 252
<212> DNA
<213> artificial sequence
<220>
<223> two copies of Xenopus beta-globin polyadenylation signal (2 xlBGpA)
<400> 13
aaccagcctc aagaacaccc gaatggagtc tctaagctac ataataccaa cttacacttt 60
acaaaatgtt gtcccccaaa atgtagccat tcgtatctgc tcctaataaa aagaaagttt 120
cttcacaacc agcctcaaga acacccgaat ggagtctcta agctacataa taccaactta 180
cactttacaa aatgttgtcc cccaaaatgt agccattcgt atctgctcct aataaaaaga 240
aagtttcttc ac 252
<210> 14
<211> 268
<212> DNA
<213> artificial sequence
<220>
<223> two copies of human beta-globin polyadenylation signal (2 huBGpA)
<400> 14
gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 60
taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt 120
tattttcatt gcaagctcgc tttcttgctg tccaatttct attaaaggtt cctttgttcc 180
ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc 240
taataaaaaa catttatttt cattgcaa 268
<210> 15
<211> 260
<212> DNA
<213> artificial sequence
<220>
<223> hybrid Xenopus and human beta-globin polyadenylation signal (xlhuBGpA)
<400> 15
aaccagcctc aagaacaccc gaatggagtc tctaagctac ataataccaa cttacacttt 60
acaaaatgtt gtcccccaaa atgtagccat tcgtatctgc tcctaataaa aagaaagttt 120
cttcacgctc gctttcttgc tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc 180
aactactaaa ctgggggata ttatgaaggg ccttgagcat ctggattctg cctaataaaa 240
aacatttatt ttcattgcaa 260
<210> 16
<211> 372
<212> DNA
<213> artificial sequence
<220>
<223> 2xlBGpA-A120
<400> 16
aaccagcctc aagaacaccc gaatggagtc tctaagctac ataataccaa cttacacttt 60
acaaaatgtt gtcccccaaa atgtagccat tcgtatctgc tcctaataaa aagaaagttt 120
cttcacaacc agcctcaaga acacccgaat ggagtctcta agctacataa taccaactta 180
cactttacaa aatgttgtcc cccaaaatgt agccattcgt atctgctcct aataaaaaga 240
aagtttcttc acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360
aaaaaaaaaa aa 372
<210> 17
<211> 388
<212> DNA
<213> artificial sequence
<220>
<223> 2huBGpA-A120
<400> 17
gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 60
taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt 120
tattttcatt gcaagctcgc tttcttgctg tccaatttct attaaaggtt cctttgttcc 180
ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc 240
taataaaaaa catttatttt cattgcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 388
<210> 18
<211> 380
<212> DNA
<213> artificial sequence
<220>
<223> xlhuBGpA-A120
<400> 18
aaccagcctc aagaacaccc gaatggagtc tctaagctac ataataccaa cttacacttt 60
acaaaatgtt gtcccccaaa atgtagccat tcgtatctgc tcctaataaa aagaaagttt 120
cttcacgctc gctttcttgc tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc 180
aactactaaa ctgggggata ttatgaaggg ccttgagcat ctggattctg cctaataaaa 240
aacatttatt ttcattgcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 360
aaaaaaaaaa aaaaaaaaaa 380
<210> 19
<211> 7011
<212> DNA
<213> artificial sequence
<220>
<223> pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*
<400> 19
ccgggaggta ccgagctctt acgcgtgcta gaattaaagt aacccaatca gcacacaatt 60
gccattatac gcgcgtataa tggactattg tgtgctgata aacctatttc agcatactac 120
gcgcgtagta tgctgaaata ggtgactaga agttcctata ctttctagag aataggaact 180
tcataacttc gtataatgta tgctatacga agttatgggt tactttaatt tggttgctga 240
ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca 300
cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg 360
ggactttcca cacccctggg tcgacattga ttattgacta gttattaata gtaatcaatt 420
acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 480
ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 540
cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 600
actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 660
aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 720
acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg 780
ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt 840
ttttaattat tttgtgcagc gatgggggcg gggggggggg gggcgcgcgc caggcggggc 900
ggggcggggc gaggggcggg gcggggcgag gcggaaaggt gcggcggcag ccaatcagag 960
cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa 1020
gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc 1080
cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg 1140
ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc tcgtttcttt 1200
tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc cctttgtgcg ggggggagcg 1260
gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc 1320
ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg 1380
aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag 1440
gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc 1500
tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1560
gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1620
ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1680
ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1740
taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1800
gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1860
aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1920
cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1980
ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 2040
ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc 2100
aaagaattga ttaattcgag cgaacgcgtc gccaccatga actccttctc cacaagcgcc 2160
ttcggtccag ttgccttctc cctgggcctg ctcctggtgt tgcctgctgc cttccctgcc 2220
ccagtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 2280
gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 2340
actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 2400
atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 2460
gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 2520
atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 2580
gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 2640
gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 2700
accggctggc ggctgtgcga acgcattctg gcggctagcg ctactaactt cagcctgctg 2760
aagcaggctg gagacgtgga ggagaaccct ggacctggaa gcggagaggg cagaggaagt 2820
ctgctaacat gcggtgacgt cgaggagaat cctggacctg gatccggaat ggtgagcaag 2880
ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 2940
ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 3000
ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 3060
ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 3120
ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 3180
ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 3240
gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 3300
aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 3360
aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 3420
cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 3480
cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 3540
gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaagc ggccgcactc 3600
ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca 3660
aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg 3720
agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 3780
ttttgtgtct ctcactcgga aggacatggt gtggaaagtc cccaggctcc ccagcaggca 3840
gaagtatgca aagcatgcat ctcaattagt cagcaaccaa attaaagtaa cccataactt 3900
cgtatagcat acattatacg aagttatgaa gttcctattc tctagaaagt ataggaactt 3960
ctagtcacct atttcagcat actacgcgcg tagtatgctg aaataggttt atcagcacac 4020
aatagtccat tatacgcgcg tataatggca attgtgtgct gattgggtta ctttaattgg 4080
tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 4140
tcagcaacca tggatccgtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt 4200
ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca 4260
actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac tcgctgcgct 4320
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 4380
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 4440
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 4500
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 4560
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 4620
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 4680
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 4740
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 4800
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 4860
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 4920
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 4980
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 5040
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 5100
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 5160
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 5220
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 5280
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 5340
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 5400
caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 5460
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 5520
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 5580
cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 5640
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 5700
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 5760
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 5820
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 5880
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 5940
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 6000
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 6060
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 6120
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 6180
taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat 6240
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6300
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6360
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6420
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6480
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6540
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6600
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 6660
taacgcttac aatttgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 6720
tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt aatattaagg 6780
tacgtggagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat 6840
aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa 6900
agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 6960
ttgtccaaac tcatcaatgt atcttatggt actgtaactg agctaacata a 7011
<210> 20
<211> 6991
<212> DNA
<213> artificial sequence
<220>
<223> pcDNA-CMV-5'UTR-SecNLuc-P2A-eGFP-bGHpA
<400> 20
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttggta ccgagctcgg atccctgcct tctccctcct gtgagtttgg 960
taagtcactg actgtctatg cctgggaaag ggtgggcagg agatggggca gtgcaggaaa 1020
agtggcacta tgaaccctgc agccctagga atgcatctag acaattgtac taaccttctt 1080
ctctttcctc tcctgacagg ttggtgtaca gtagcttcca ctcctgccac catgaactcc 1140
ttctccacaa gcgccttcgg tccagttgcc ttctccctgg gcctgctcct ggtgttgcct 1200
gctgccttcc ctgccccagt cttcacactc gaagatttcg ttggggactg gcgacagaca 1260
gccggctaca acctggacca agtccttgaa cagggaggtg tgtccagttt gtttcagaat 1320
ctcggggtgt ccgtaactcc gatccaaagg attgtcctga gcggtgaaaa tgggctgaag 1380
atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg gcgaccaaat gggccagatc 1440
gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc actttaaggt gatcctgcac 1500
tatggcacac tggtaatcga cggggttacg ccgaacatga tcgactattt cggacggccg 1560
tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg taacagggac cctgtggaac 1620
ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg gctccctgct gttccgagta 1680
accatcaacg gagtgaccgg ctggcggctg tgcgaacgca ttctggcgga attctgcaga 1740
tatccagcac agtggcggcc gctcgagtct agaggaagcg gagctactaa cttcagcctg 1800
ctgaagcagg ctggagacgt ggaggagaac cctggaccta tgagcaaggg cgaggagctg 1860
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 1920
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 1980
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 2040
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2100
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2160
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2220
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2280
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2340
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 2400
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 2460
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 2520
gggatcactc acggcatgga cgagctgtac aagtaagggc ccgtttaaac ccgctgatca 2580
gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2640
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2700
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2760
gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 2820
gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 2880
agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 2940
cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 3000
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 3060
aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 3120
cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 3180
acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 3240
tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 3300
tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 3360
tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 3420
gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 3480
tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 3540
ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 3600
gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 3660
gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 3720
caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 3780
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 3840
tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 3900
ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 3960
gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 4020
ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 4080
ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 4140
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 4200
aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 4260
gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 4320
gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 4380
ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 4440
ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 4500
ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 4560
cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 4620
cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 4680
ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 4740
actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 4800
gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 4860
ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 4920
tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 4980
gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 5040
gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 5100
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 5160
taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 5220
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 5280
ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 5340
aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 5400
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 5460
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 5520
cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 5580
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 5640
cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 5700
gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 5760
cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 5820
tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 5880
ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 5940
aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 6000
atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 6060
ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 6120
tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 6180
agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 6240
taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 6300
tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 6360
cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 6420
ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 6480
tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 6540
tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 6600
cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 6660
tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 6720
gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 6780
tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 6840
atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 6900
tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 6960
cacatttccc cgaaaagtgc cacctgacgt c 6991
<210> 21
<211> 4555
<212> DNA
<213> artificial sequence
<220>
<223> pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS*
<400> 21
taaagtaacc caatcagcac acaattgcca ttatacgcgc gtataatgga ctattgtgtg 60
ctgataaacc tatttcagca tactacgcgc gtagtatgct gaaataggtg actagaagtt 120
cctatacttt ctagagaata ggaacttcat aacttcgtat aatgtatgct atacgaagtt 180
atgggttact ttaatttggt tgctgactaa ttgagatgca tgctttgcat acttctgcct 240
gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc 300
atacttctgc ctgctgggga gcctggggac tttccacacc cctgggtcga cattgattat 360
tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 420
tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 480
cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 540
gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 600
tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 660
agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 720
ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 780
cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 840
gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 900
aaaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 960
cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgttg 1020
ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc cgccccggct ctgactgacc 1080
gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc 1140
ttggtttaat gacggctcgt ttcttttctg tggctgcgtg aaagccttaa agggctccgg 1200
gagggccctt tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg 1260
gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg 1320
gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg 1380
cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc 1440
agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg 1500
ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg 1560
tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg 1620
gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg 1680
cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt 1740
cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg 1800
gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc 1860
cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc 1920
ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 1980
cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 2040
ttattgtgct gtctcatcat tttggcaaag aattgattaa ttcgagcgaa cgcgtcgcca 2100
ccatgaactc cttctccaca agcgccttcg gtccagttgc cttctccctg ggcctgctcc 2160
tggtgttgcc tgctgccttc cctgccccag tcttcacact cgaagatttc gttggggact 2220
ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt 2280
tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa 2340
atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa 2400
tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg 2460
tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt 2520
tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga 2580
ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc 2640
tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggcgg 2700
ctagcgctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac 2760
ctggaagcgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg 2820
gacctggatc cggaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 2880
tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 2940
gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 3000
tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 3060
ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 3120
agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 3180
agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 3240
acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 3300
acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 3360
gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 3420
tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc 3480
gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 3540
agctgtacaa gtaaaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 3600
ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 3660
ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc 3720
tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg 3780
acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg 3840
ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 3900
caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct 3960
ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg 4020
tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc 4080
ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc 4140
cgcaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac 4200
tcggaaggac atggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 4260
tgcatctcaa ttagtcagca accaaattaa agtaacccat aacttcgtat agcatacatt 4320
atacgaagtt atgaagttcc tattctctag aaagtatagg aacttctagt cacctatttc 4380
agcatactac gcgcgtagta tgctgaaata ggtttatcag cacacaatag tccattatac 4440
gcgcgtataa tggcaattgt gtgctgattg ggttacttta attggtgtgg aaagtcccca 4500
ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aacca 4555
<210> 22
<211> 3610
<212> DNA
<213> artificial sequence
<220>
<223> pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*
<400> 22
taaagtaacc caatcagcac acaattgcca ttatacgcgc gtataatgga ctattgtgtg 60
ctgataaacc tatttcagca tactacgcgc gtagtatgct gaaataggtg actagaagtt 120
cctatacttt ctagagaata ggaacttcat aacttcgtat aatgtatgct atacgaagtt 180
atgggttact ttaatttggt tgctgactaa ttgagatgca tgctttgcat acttctgcct 240
gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc 300
atacttctgc ctgctgggga gcctggggac tttccacacc cctgggtcga cgacattgat 360
tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 420
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 480
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 540
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 600
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 660
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 720
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 780
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 840
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 900
ggcgtgtacg gtgggaggtc tatataagca gagctctgcc ttctccctcc tgtgagtttg 960
gtaagtcgac gggccgggcc tgggccgggt ccgggccggg tcgttggatc cccactacag 1020
cccgatactc aagcttgacg aattcgagta tccaaggtag tggactagtg tgacgctgct 1080
gacccctttc tttcccttct gcaggttggt gtacagtagc ttccaaattg attaattcga 1140
gcgaacgcgt cgccaccatg aactccttct ccacaagcgc cttcggtcca gttgccttct 1200
ccctgggcct gctcctggtg ttgcctgctg ccttccctgc cccagtcttc acactcgaag 1260
atttcgttgg ggactggcga cagacagccg gctacaacct ggaccaagtc cttgaacagg 1320
gaggtgtgtc cagtttgttt cagaatctcg gggtgtccgt aactccgatc caaaggattg 1380
tcctgagcgg tgaaaatggg ctgaagatcg acatccatgt catcatcccg tatgaaggtc 1440
tgagcggcga ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg 1500
atcatcactt taaggtgatc ctgcactatg gcacactggt aatcgacggg gttacgccga 1560
acatgatcga ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga 1620
tcactgtaac agggaccctg tggaacggca acaaaattat cgacgagcgc ctgatcaacc 1680
ccgacggctc cctgctgttc cgagtaacca tcaacggagt gaccggctgg cggctgtgcg 1740
aacgcattct ggcggctagc gctactaact tcagcctgct gaagcaggct ggagacgtgg 1800
aggagaaccc tggacctgga agcggagagg gcagaggaag tctgctaaca tgcggtgacg 1860
tcgaggagaa tcctggacct ggatccggaa tggtgagcaa gggcgaggag ctgttcaccg 1920
gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 1980
ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 2040
ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 2100
gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 2160
aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 2220
ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 2280
tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 2340
tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 2400
acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 2460
acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 2520
accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 2580
ctctcggcat ggacgagctg tacaagtaaa atcaacctct ggattacaaa atttgtgaaa 2640
gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 2700
tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 2760
cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 2820
gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 2880
tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 2940
ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 3000
ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 3060
cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 3120
tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 3180
tttgggccgc ctccccgcaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt 3240
ttttgtgtct ctcactcgga aggacatggt gtggaaagtc cccaggctcc ccagcaggca 3300
gaagtatgca aagcatgcat ctcaattagt cagcaaccaa attaaagtaa cccataactt 3360
cgtatagcat acattatacg aagttatgaa gttcctattc tctagaaagt ataggaactt 3420
ctagtcacct atttcagcat actacgcgcg tagtatgctg aaataggttt atcagcacac 3480
aatagtccat tatacgcgcg tataatggca attgtgtgct gattgggtta ctttaattgg 3540
tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 3600
tcagcaacca 3610
<210> 23
<211> 4092
<212> DNA
<213> artificial sequence
<220>
<223> pGL2-SS*-CMV-UTR2-SecNLuc-2A-eGFP-WPRE-BGpA-SS*
<400> 23
aattaaagta acccaatcag cacacaattg ccattatacg cgcgtataat ggactattgt 60
gtgctgataa acctatttca gcatactacg cgcgtagtat gctgaaatag gtgactagaa 120
gttcctatac tttctagaga ataggaactt cataacttcg tataatgtat gctatacgaa 180
gttatgggtt actttaattt ggttgctgac taattgagat gcatgctttg catacttctg 240
cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt 300
tgcatacttc tgcctgctgg ggagcctggg gactttccac acccctgggt cgacattgat 360
tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg 420
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 480
gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt 540
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 600
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 660
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 720
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 780
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 840
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 900
ggcgtgtacg gtgggaggtc tatataagca gagctattgg gatcttcaca cagcaggtaa 960
ggttgcgggc cgggcctggg ccgggtccgg gccgggtatc catagctgat tggtctaaaa 1020
tgagatacat caacgctcct ccatgttttt tgttttcttt ttaaatgaaa aactttattt 1080
tttaagagga gtttcaggtt catagcaaaa ttgagaggaa ggtacattca agctgaggaa 1140
gttttcctct attcctagtt tactgagaga ttgcatcatg aatgggtgtt aaattttgtc 1200
aaatgctttt tctgtgtcta tcaatatgac catgtgattt tcttctttaa cctgttgatg 1260
ggacaaatta cgttaattga ttttcaaacg ttgaaccacc cttacatatc tggaataaat 1320
tctacttggt tgtggtgtat attttttgat acattcttgg attctttttg ctaatatttt 1380
gttgaaaatg tttgtatctt tgttcatgag agatattggt ctgttgtttt cttttcttgt 1440
aatgtcattt tctagttccg gtattaaggt aatgctggcc tagttgaatg atttaggaag 1500
tattccctct gcttctgtct tctgaaagag attgtagaaa gttgatacaa tttttttttc 1560
tttaaatatc ttgatagccg cactgacccc tggtgttgct tttttttttt aggccgcaag 1620
ctgaagcgtg tccgccacca tgaactcctt ctccacaagc gccttcggtc cagttgcctt 1680
ctccctgggc ctgctcctgg tgttgcctgc tgccttccct gccccagtct tcacactcga 1740
agatttcgtt ggggactggc gacagacagc cggctacaac ctggaccaag tccttgaaca 1800
gggaggtgtg tccagtttgt ttcagaatct cggggtgtcc gtaactccga tccaaaggat 1860
tgtcctgagc ggtgaaaatg ggctgaagat cgacatccat gtcatcatcc cgtatgaagg 1920
tctgagcggc gaccaaatgg gccagatcga aaaaattttt aaggtggtgt accctgtgga 1980
tgatcatcac tttaaggtga tcctgcacta tggcacactg gtaatcgacg gggttacgcc 2040
gaacatgatc gactatttcg gacggccgta tgaaggcatc gccgtgttcg acggcaaaaa 2100
gatcactgta acagggaccc tgtggaacgg caacaaaatt atcgacgagc gcctgatcaa 2160
ccccgacggc tccctgctgt tccgagtaac catcaacgga gtgaccggct ggcggctgtg 2220
cgaacgcatt ctggcggcta gcgctactaa cttcagcctg ctgaagcagg ctggagacgt 2280
ggaggagaac cctggacctg gaagcggaga gggcagagga agtctgctaa catgcggtga 2340
cgtcgaggag aatcctggac ctggatccgg aatggtgagc aagggcgagg agctgttcac 2400
cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 2460
gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 2520
caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 2580
gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 2640
cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 2700
cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 2760
cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 2820
cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 2880
caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 2940
cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 3000
agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 3060
cactctcggc atggacgagc tgtacaagta aaatcaacct ctggattaca aaatttgtga 3120
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 3180
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 3240
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 3300
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 3360
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 3420
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 3480
ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 3540
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 3600
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 3660
cctttgggcc gcctccccgc aataaaggaa atttattttc attgcaatag tgtgttggaa 3720
ttttttgtgt ctctcactcg gaaggacatg gtgtggaaag tccccaggct ccccagcagg 3780
cagaagtatg caaagcatgc atctcaatta gtcagcaacc aaattaaagt aacccataac 3840
ttcgtatagc atacattata cgaagttatg aagttcctat tctctagaaa gtataggaac 3900
ttctagtcac ctatttcagc atactacgcg cgtagtatgc tgaaataggt ttatcagcac 3960
acaatagtcc attatacgcg cgtataatgg caattgtgtg ctgattgggt tactttaatt 4020
ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 4080
agtcagcaac ca 4092
<210> 24
<211> 6249
<212> DNA
<213> artificial sequence
<220>
<223> SS*-CMV-UTR1-SecNLuc-2A-eGFP-3'UTR[2huBGpA-A120]-SS*
<400> 24
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgaca ttgattattg actagttatt aatagtaatc 420
aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 480
aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 540
tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 600
gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 660
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 720
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 780
gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 840
cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 900
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 960
aagcagagct ctgccttctc cctcctgtga gtttggtaag tcgacgggcc gggcctgggc 1020
cgggtccggg ccgggtcgtt ggatccccac tacagcccga tactcaagct tgacgaattc 1080
gagtatccaa ggtagtggac tagtgtgacg ctgctgaccc ctttctttcc cttctgcagg 1140
ttggtgtaca gtagcttcca aattgattaa ttcgagcgaa cgcgtcgcca ccatgaactc 1200
cttctccaca agcgccttcg gtccagttgc cttctccctg ggcctgctcc tggtgttgcc 1260
tgctgccttc cctgccccag tcttcacact cgaagatttc gttggggact ggcgacagac 1320
agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt tgtttcagaa 1380
tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa atgggctgaa 1440
gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa tgggccagat 1500
cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg tgatcctgca 1560
ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt tcggacggcc 1620
gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga ccctgtggaa 1680
cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc tgttccgagt 1740
aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggcgg ctagcgctac 1800
taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac ctggaagcgg 1860
agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gacctggatc 1920
cggaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 1980
ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 2040
ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 2100
caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 2160
gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 2220
cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 2280
cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 2340
gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 2400
gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 2460
cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 2520
ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 2580
ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 2640
gtaagctcgc tttcttgctg tccaatttct attaaaggtt cctttgttcc ctaagtccaa 2700
ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc taataaaaaa 2760
catttatttt cattgcaagc tcgctttctt gctgtccaat ttctattaaa ggttcctttg 2820
ttccctaagt ccaactacta aactggggga tattatgaag ggccttgagc atctggattc 2880
tgcctaataa aaaacattta ttttcattgc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2940
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aactcggaag gacatggtgt ggaaagtccc 3060
caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt 3120
gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt 3180
cagcaaccaa attaaagtaa cccataactt cgtatagcat acattatacg aagttatgaa 3240
gttcctattc tctagaaagt ataggaactt ctagtcacct atttcagcat actacgcgcg 3300
tagtatgctg aaataggttt atcagcacac aatagtccat tatacgcgcg tataatggca 3360
attgtgtgct gattgggtta ctttaatttg gatccgtcga ccgatgccct tgagagcctt 3420
caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac 3480
tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc 3540
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3600
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3660
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3720
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3780
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3840
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3900
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3960
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4020
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4080
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4140
ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4200
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4260
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4320
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4380
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4440
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4500
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4560
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 4620
cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4680
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4740
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4800
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4860
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4920
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4980
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 5040
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 5100
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 5160
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 5220
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 5280
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5340
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5400
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5460
cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 5520
cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 5580
tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 5640
ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 5700
cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 5760
tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 5820
ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 5880
cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact 5940
gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat 6000
aagtaagtaa tattaaggta cgtggaggtt ttacttgctt taaaaaacct cccacacctc 6060
cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct 6120
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 6180
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatggtac tgtaactgag 6240
ctaacataa 6249
<210> 25
<211> 6360
<212> DNA
<213> artificial sequence
<220>
<223> SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-3'UTR[2huBGpA-A120]-SS*
<400> 25
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacggga ctttccgggg cggggcacgt ggtgcacggg 420
actttccgtg cacgtgcacg ggactttccg ggactttccg ggactttccg tgcaccacgt 480
ggggactttc cgtgcacgac attgattatt gactagttat taatagtaat caattacggg 540
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 600
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 660
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 720
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 780
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 840
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 900
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 960
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 1020
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 1080
tctgccttct ccctcctgtg agtttggtaa gtcgacgggc cgggcctggg ccgggtccgg 1140
gccgggtcgt tggatcccca ctacagcccg atactcaagc ttgacgaatt cgagtatcca 1200
aggtagtgga ctagtgtgac gctgctgacc cctttctttc ccttctgcag gttggtgtac 1260
agtagcttcc aaattgatta attcgagcga acgcgtcgcc accatgaact ccttctccac 1320
aagcgccttc ggtccagttg ccttctccct gggcctgctc ctggtgttgc ctgctgcctt 1380
ccctgcccca gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta 1440
caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt 1500
gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat 1560
ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat 1620
ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac 1680
actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg 1740
catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa 1800
aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa 1860
cggagtgacc ggctggcggc tgtgcgaacg cattctggcg gctagcgcta ctaacttcag 1920
cctgctgaag caggctggag acgtggagga gaaccctgga cctggaagcg gagagggcag 1980
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat ccggaatggt 2040
gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 2100
cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa 2160
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 2220
gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 2280
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 2340
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 2400
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 2460
ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 2520
caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 2580
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 2640
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 2700
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaagctcg 2760
ctttcttgct gtccaatttc tattaaaggt tcctttgttc cctaagtcca actactaaac 2820
tgggggatat tatgaagggc cttgagcatc tggattctgc ctaataaaaa acatttattt 2880
tcattgcaag ctcgctttct tgctgtccaa tttctattaa aggttccttt gttccctaag 2940
tccaactact aaactggggg atattatgaa gggccttgag catctggatt ctgcctaata 3000
aaaaacattt attttcattg caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3060
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120
aaaaaaaaaa aaaaaaaaaa aaactcggaa ggacatggtg tggaaagtcc ccaggctccc 3180
cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 3240
ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 3300
aattaaagta acccataact tcgtatagca tacattatac gaagttatga agttcctatt 3360
ctctagaaag tataggaact tctagtcacc tatttcagca tactacgcgc gtagtatgct 3420
gaaataggtt tatcagcaca caatagtcca ttatacgcgc gtataatggc aattgtgtgc 3480
tgattgggtt actttaattt ggatccgtcg accgatgccc ttgagagcct tcaacccagt 3540
cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt 3600
tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg ctcactgact 3660
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3720
ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3780
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3840
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3900
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 3960
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 4020
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4080
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4140
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4200
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 4260
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4320
cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 4380
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 4440
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 4500
tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 4560
aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 4620
tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 4680
gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 4740
atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4800
tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4860
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 4920
ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 4980
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 5040
ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 5100
ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 5160
tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 5220
gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5280
taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5340
cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5400
agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 5460
gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 5520
ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcgccctgta 5580
gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 5640
gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 5700
ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 5760
acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 5820
agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 5880
aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 5940
cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 6000
acaaaatatt aacgcttaca atttgccatt cgccattcag gctgcgcaac tgttgggaag 6060
ggcgatcggt gcgggcctct tcgctattac gccagcccaa gctaccatga taagtaagta 6120
atattaaggt acgtggaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 6180
ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 6240
tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 6300
agttgtggtt tgtccaaact catcaatgta tcttatggta ctgtaactga gctaacataa 6360
<210> 26
<211> 6949
<212> DNA
<213> artificial sequence
<220>
<223> SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-3'UTR[2huBGpA-A120]-SS*
<400> 26
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacggga ctttccgggg cggggcacgt ggtgcacggg 420
actttccgtg cacgtgcacg ggactttccg ggactttccg ggactttccg tgcaccacgt 480
ggggactttc cgtgcacgac attgattatt gactagttat taatagtaat caattacggg 540
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 600
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 660
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 720
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 780
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 840
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 900
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 960
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 1020
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 1080
tctgccttct ccctcctgtg agtttggtaa gtcgacgggc cgggcctggg ccgggtccgg 1140
gccgggtcgt tggatcccca ctacagcccg atactcaagc ttgacgaatt cgagtatcca 1200
aggtagtgga ctagtgtgac gctgctgacc cctttctttc ccttctgcag gttggtgtac 1260
agtagcttcc aaattgatta attcgagcga acgcgtcgcc accatgaact ccttctccac 1320
aagcgccttc ggtccagttg ccttctccct gggcctgctc ctggtgttgc ctgctgcctt 1380
ccctgcccca gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta 1440
caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt 1500
gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat 1560
ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat 1620
ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac 1680
actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg 1740
catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa 1800
aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa 1860
cggagtgacc ggctggcggc tgtgcgaacg cattctggcg gctagcgcta ctaacttcag 1920
cctgctgaag caggctggag acgtggagga gaaccctgga cctggaagcg gagagggcag 1980
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat ccggaatggt 2040
gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 2100
cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa 2160
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 2220
gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 2280
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 2340
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 2400
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 2460
ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 2520
caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 2580
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 2640
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 2700
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaaatca 2760
acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 2820
tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 2880
tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 2940
cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 3000
gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 3060
cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 3120
cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg 3180
tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 3240
agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 3300
tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgctcgc tttcttgctg 3360
tccaatttct attaaaggtt cctttgttcc ctaagtccaa ctactaaact gggggatatt 3420
atgaagggcc ttgagcatct ggattctgcc taataaaaaa catttatttt cattgcaagc 3480
tcgctttctt gctgtccaat ttctattaaa ggttcctttg ttccctaagt ccaactacta 3540
aactggggga tattatgaag ggccttgagc atctggattc tgcctaataa aaaacattta 3600
ttttcattgc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3720
aaaaaaaaaa aactcggaag gacatggtgt ggaaagtccc caggctcccc agcaggcaga 3780
agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 3840
ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccaa attaaagtaa 3900
cccataactt cgtatagcat acattatacg aagttatgaa gttcctattc tctagaaagt 3960
ataggaactt ctagtcacct atttcagcat actacgcgcg tagtatgctg aaataggttt 4020
atcagcacac aatagtccat tatacgcgcg tataatggca attgtgtgct gattgggtta 4080
ctttaatttg gatccgtcga ccgatgccct tgagagcctt caacccagtc agctccttcc 4140
ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 4200
tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4260
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 4320
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 4380
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 4440
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 4500
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 4560
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 4620
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 4680
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 4740
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 4800
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 4860
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 4920
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 4980
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5040
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5100
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5160
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 5220
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 5280
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 5340
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 5400
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 5460
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 5520
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 5580
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 5640
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 5700
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 5760
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 5820
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 5880
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 5940
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 6000
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 6060
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 6120
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag cggcgcatta 6180
agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 6240
cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 6300
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 6360
aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 6420
cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 6480
acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 6540
tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 6600
acgcttacaa tttgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 6660
cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa tattaaggta 6720
cgtggaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa 6780
aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag 6840
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 6900
gtccaaactc atcaatgtat cttatggtac tgtaactgag ctaacataa 6949
<210> 27
<211> 7121
<212> DNA
<213> artificial sequence
<220>
<223> SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-3'UTR[2huBGpA-A120]-SS*
<400> 27
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgcac acgaccacaa ttccactgaa agcattttaa 420
tacggaactt gtcactccca gggagcctcc gctcagccgg cagttggttc atttcaatcc 480
ccacgacaac ccttcaaagt gcagggcaga cagcaggtgg ctctgcccag gcgcctggat 540
cacagcccgg cctgcagccc tcacctgggc gcggggagac cctgaggacg ctcctccagg 600
cggcgctggc cggggcctgc ggacacggac gggcgggctg agctccggga cccctccccg 660
cgccccgcac cccgcacccc gcaccccgca ccccgcaccc ggcgctcacc cgtcccagcc 720
ccgccgcccg cagccccagc tgcaacgcag ccaccgccgc catcgcaccc ggccccgcgg 780
gcgcttccgg gacgcaggag gcatctgcat ccggggcgcc gctgagtccc gcccagagcc 840
ccgcccccgg ctccaggttc tgcgagcggc ttccgccggg ctgctccgcg ggcgcgtcgg 900
ccatgagcga gttgccgggc gacgtgcggg cgtttctgcg ggagcacccg agcctgcggc 960
tccagacgga cgcccgcaag gttcgcagcg cgggagggga acggagtggc ggagaagggc 1020
gcagttggga tgaggggctg aggggagggc aggggagagg agagggcagg ggagagggga 1080
gaggggagag caggagagag gggaaggcag gggagagggc gcggcgggat caggggagga 1140
gagggaaggg actttccggg gcggggcacg tggtgcacgg gactttccgt gcacgtgcac 1200
gggactttcc gggactttcc gggactttcc gtgcaccacg tggggacttt ccgtgcacga 1260
cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 1320
tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 1380
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 1440
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1500
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1560
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1620
gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 1680
tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 1740
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 1800
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctgccttc tccctcctgt 1860
gagtttggta agtcgacggg ccgggcctgg gccgggtccg ggccgggtcg ttggatcccc 1920
actacagccc gatactcaag cttgacgaat tcgagtatcc aaggtagtgg actagtgtga 1980
cgctgctgac ccctttcttt cccttctgca ggttggtgta cagtagcttc caaattgatt 2040
aattcgagcg aacgcgtcgc caccatgaac tccttctcca caagcgcctt cggtccagtt 2100
gccttctccc tgggcctgct cctggtgttg cctgctgcct tccctgcccc agtcttcaca 2160
ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt 2220
gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa 2280
aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat 2340
gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct 2400
gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt 2460
acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc 2520
aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg 2580
atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg 2640
ctgtgcgaac gcattctggc ggctagcgct actaacttca gcctgctgaa gcaggctgga 2700
gacgtggagg agaaccctgg acctggaagc ggagagggca gaggaagtct gctaacatgc 2760
ggtgacgtcg aggagaatcc tggacctgga tccggaatgg tgagcaaggg cgaggagctg 2820
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2880
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 2940
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 3000
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 3060
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 3120
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 3180
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 3240
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 3300
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 3360
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 3420
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3480
gggatcactc tcggcatgga cgagctgtac aagtaagctc gctttcttgc tgtccaattt 3540
ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata ttatgaaggg 3600
ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa gctcgctttc 3660
ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac taaactgggg 3720
gatattatga agggccttga gcatctggat tctgcctaat aaaaaacatt tattttcatt 3780
gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3900
aaaactcgga aggacatggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3960
aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg 4020
cagaagtatg caaagcatgc atctcaatta gtcagcaacc aaattaaagt aacccataac 4080
ttcgtatagc atacattata cgaagttatg aagttcctat tctctagaaa gtataggaac 4140
ttctagtcac ctatttcagc atactacgcg cgtagtatgc tgaaataggt ttatcagcac 4200
acaatagtcc attatacgcg cgtataatgg caattgtgtg ctgattgggt tactttaatt 4260
tggatccgtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg 4320
cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga 4380
caggtgccgg cagcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4440
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4500
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4560
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4620
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4680
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4740
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4800
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4860
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4920
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4980
gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 5040
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5100
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5160
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5220
acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5280
ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5340
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5400
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5460
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5520
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5580
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5640
tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5700
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5760
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5820
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5880
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 5940
ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6000
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6060
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6120
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6180
gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6240
ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6300
gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc 6360
gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 6420
tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 6480
tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 6540
tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 6600
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 6660
ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 6720
aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac 6780
aatttgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 6840
ttcgctatta cgccagccca agctaccatg ataagtaagt aatattaagg tacgtggagg 6900
ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 6960
caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 7020
tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 7080
tcatcaatgt atcttatggt actgtaactg agctaacata a 7121
<210> 28
<211> 7593
<212> DNA
<213> artificial sequence
<220>
<223> SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-huMAR-3'UTR[2huBGpA-A120]-SS*
<400> 28
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacggga ctttccgggg cggggcacgt ggtgcacggg 420
actttccgtg cacgtgcacg ggactttccg ggactttccg ggactttccg tgcaccacgt 480
ggggactttc cgtgcacgac attgattatt gactagttat taatagtaat caattacggg 540
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 600
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 660
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 720
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 780
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 840
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 900
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 960
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 1020
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 1080
tctgccttct ccctcctgtg agtttggtaa gtcgacgggc cgggcctggg ccgggtccgg 1140
gccgggtcgt tggatcccca ctacagcccg atactcaagc ttgacgaatt cgagtatcca 1200
aggtagtgga ctagtgtgac gctgctgacc cctttctttc ccttctgcag gttggtgtac 1260
agtagcttcc aaattgatta attcgagcga acgcgtcgcc accatgaact ccttctccac 1320
aagcgccttc ggtccagttg ccttctccct gggcctgctc ctggtgttgc ctgctgcctt 1380
ccctgcccca gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta 1440
caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt 1500
gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat 1560
ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat 1620
ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac 1680
actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg 1740
catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa 1800
aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa 1860
cggagtgacc ggctggcggc tgtgcgaacg cattctggcg gctagcgcta ctaacttcag 1920
cctgctgaag caggctggag acgtggagga gaaccctgga cctggaagcg gagagggcag 1980
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat ccggaatggt 2040
gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 2100
cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa 2160
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 2220
gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 2280
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 2340
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 2400
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 2460
ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 2520
caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 2580
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 2640
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 2700
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaggatc 2760
ccattctcct tgatgtacta atttttcttt aaaagtgata ataatagctc ccatttagaa 2820
tttttaaata acacaacaaa tgtaaagtaa ctaatgtgtc ctctggatca tggtaagtaa 2880
tgaataaatt taactccctt taccttctcc ctttgctatt ttttccatgc taggatttat 2940
acatttttaa aaaactaaat ctgctatcaa atgacagctt taaatttact ttttaaaatt 3000
tgttattgta tatatttatg gggtataaag tgatgttatg atatatatat acacaatgta 3060
cactgattaa atcaagccaa ttaacatttt atcatctcaa atacttaaca ttttttgtag 3120
tgagaacatt tgaaatttac ttttagcaat ttcaaaacat acattattat tattaactat 3180
agtcaccatg atgtaccata gatctttaaa aacttattct tcctgcctaa ctgaaacttt 3240
gtactctttg actaacatct tttcattccc ccacttccca gcctctggta atcaccatta 3300
cacactctgc ttctatgagt tcaattgctt tagactccac gtaataaatg agatcatgca 3360
gcatttggct ttctgtgcct ggcttatcct tgcttagcat ggtgtcttac aggttcatcc 3420
atgttgcaac aaataacaga atctcattct ttgttaaggc tgaatactat tccattgggt 3480
atatatacca cattttcctt atccattaat ccactgatgg acccttaggt tgttgattcc 3540
atatattggc tattgtaaat agtgcagcaa tgaacatgag agtgcaacta tctcttcaat 3600
gtactgattt cgaatccttc ggatctatct cagaagtgag attgcaggat catataattc 3660
tacttttagt cttttgagga gctccataca gctttccata tggccatact aattacattc 3720
tcatcaacag tgtacaatgg tttccttttc tccacatcct caccaacatt tataattttt 3780
tgtctttttg ataatagcca tctgacaggt gtaaagtgat agctcattgc agttttaatt 3840
tgcatttttt gatgattagt aatgttgaga attttttcat atatctcttg gccagttgca 3900
tgtcttcttt ggaaaaatgt ctattcagtt cctttgccca ttttttaatt gggatttttg 3960
gtttcttgct attgagttgt ttgaattcgc tcgctttctt gctgtccaat ttctattaaa 4020
ggttcctttg ttccctaagt ccaactacta aactggggga tattatgaag ggccttgagc 4080
atctggattc tgcctaataa aaaacattta ttttcattgc aagctcgctt tcttgctgtc 4140
caatttctat taaaggttcc tttgttccct aagtccaact actaaactgg gggatattat 4200
gaagggcctt gagcatctgg attctgccta ataaaaaaca tttattttca ttgcaaaaaa 4260
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4320
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaactcg 4380
gaaggacatg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 4440
atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta 4500
tgcaaagcat gcatctcaat tagtcagcaa ccaaattaaa gtaacccata acttcgtata 4560
gcatacatta tacgaagtta tgaagttcct attctctaga aagtatagga acttctagtc 4620
acctatttca gcatactacg cgcgtagtat gctgaaatag gtttatcagc acacaatagt 4680
ccattatacg cgcgtataat ggcaattgtg tgctgattgg gttactttaa tttggatccg 4740
tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat 4800
gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc 4860
ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 4920
gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 4980
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 5040
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 5100
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5160
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5220
cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 5280
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5340
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5400
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5460
agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 5520
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 5580
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 5640
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 5700
ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 5760
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 5820
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 5880
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 5940
tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 6000
gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 6060
gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 6120
ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 6180
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6240
tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 6300
cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 6360
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 6420
cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 6480
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 6540
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 6600
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 6660
gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 6720
tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 6780
ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6840
tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 6900
tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6960
tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 7020
gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 7080
agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 7140
cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 7200
agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttgcc 7260
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 7320
tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgtgga ggttttactt 7380
gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 7440
gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 7500
ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 7560
gtatcttatg gtactgtaac tgagctaaca taa 7593
<210> 29
<211> 8354
<212> DNA
<213> artificial sequence
<220>
<223> SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-huMAR-3'UTR[2huBGpA-A120]-SS
*
<400> 29
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgcac acgaccacaa ttccactgaa agcattttaa 420
tacggaactt gtcactccca gggagcctcc gctcagccgg cagttggttc atttcaatcc 480
ccacgacaac ccttcaaagt gcagggcaga cagcaggtgg ctctgcccag gcgcctggat 540
cacagcccgg cctgcagccc tcacctgggc gcggggagac cctgaggacg ctcctccagg 600
cggcgctggc cggggcctgc ggacacggac gggcgggctg agctccggga cccctccccg 660
cgccccgcac cccgcacccc gcaccccgca ccccgcaccc ggcgctcacc cgtcccagcc 720
ccgccgcccg cagccccagc tgcaacgcag ccaccgccgc catcgcaccc ggccccgcgg 780
gcgcttccgg gacgcaggag gcatctgcat ccggggcgcc gctgagtccc gcccagagcc 840
ccgcccccgg ctccaggttc tgcgagcggc ttccgccggg ctgctccgcg ggcgcgtcgg 900
ccatgagcga gttgccgggc gacgtgcggg cgtttctgcg ggagcacccg agcctgcggc 960
tccagacgga cgcccgcaag gttcgcagcg cgggagggga acggagtggc ggagaagggc 1020
gcagttggga tgaggggctg aggggagggc aggggagagg agagggcagg ggagagggga 1080
gaggggagag caggagagag gggaaggcag gggagagggc gcggcgggat caggggagga 1140
gagggaaggg actttccggg gcggggcacg tggtgcacgg gactttccgt gcacgtgcac 1200
gggactttcc gggactttcc gggactttcc gtgcaccacg tggggacttt ccgtgcacga 1260
cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 1320
tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 1380
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 1440
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1500
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1560
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1620
gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 1680
tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 1740
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 1800
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctgccttc tccctcctgt 1860
gagtttggta agtcgacggg ccgggcctgg gccgggtccg ggccgggtcg ttggatcccc 1920
actacagccc gatactcaag cttgacgaat tcgagtatcc aaggtagtgg actagtgtga 1980
cgctgctgac ccctttcttt cccttctgca ggttggtgta cagtagcttc caaattgatt 2040
aattcgagcg aacgcgtcgc caccatgaac tccttctcca caagcgcctt cggtccagtt 2100
gccttctccc tgggcctgct cctggtgttg cctgctgcct tccctgcccc agtcttcaca 2160
ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt 2220
gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa 2280
aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat 2340
gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct 2400
gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt 2460
acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc 2520
aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg 2580
atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg 2640
ctgtgcgaac gcattctggc ggctagcgct actaacttca gcctgctgaa gcaggctgga 2700
gacgtggagg agaaccctgg acctggaagc ggagagggca gaggaagtct gctaacatgc 2760
ggtgacgtcg aggagaatcc tggacctgga tccggaatgg tgagcaaggg cgaggagctg 2820
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2880
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 2940
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 3000
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 3060
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 3120
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 3180
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 3240
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 3300
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 3360
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 3420
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3480
gggatcactc tcggcatgga cgagctgtac aagtaaggat cccattctcc ttgatgtact 3540
aatttttctt taaaagtgat aataatagct cccatttaga atttttaaat aacacaacaa 3600
atgtaaagta actaatgtgt cctctggatc atggtaagta atgaataaat ttaactccct 3660
ttaccttctc cctttgctat tttttccatg ctaggattta tacattttta aaaaactaaa 3720
tctgctatca aatgacagct ttaaatttac tttttaaaat ttgttattgt atatatttat 3780
ggggtataaa gtgatgttat gatatatata tacacaatgt acactgatta aatcaagcca 3840
attaacattt tatcatctca aatacttaac attttttgta gtgagaacat ttgaaattta 3900
cttttagcaa tttcaaaaca tacattatta ttattaacta tagtcaccat gatgtaccat 3960
agatctttaa aaacttattc ttcctgccta actgaaactt tgtactcttt gactaacatc 4020
ttttcattcc cccacttccc agcctctggt aatcaccatt acacactctg cttctatgag 4080
ttcaattgct ttagactcca cgtaataaat gagatcatgc agcatttggc tttctgtgcc 4140
tggcttatcc ttgcttagca tggtgtctta caggttcatc catgttgcaa caaataacag 4200
aatctcattc tttgttaagg ctgaatacta ttccattggg tatatatacc acattttcct 4260
tatccattaa tccactgatg gacccttagg ttgttgattc catatattgg ctattgtaaa 4320
tagtgcagca atgaacatga gagtgcaact atctcttcaa tgtactgatt tcgaatcctt 4380
cggatctatc tcagaagtga gattgcagga tcatataatt ctacttttag tcttttgagg 4440
agctccatac agctttccat atggccatac taattacatt ctcatcaaca gtgtacaatg 4500
gtttcctttt ctccacatcc tcaccaacat ttataatttt ttgtcttttt gataatagcc 4560
atctgacagg tgtaaagtga tagctcattg cagttttaat ttgcattttt tgatgattag 4620
taatgttgag aattttttca tatatctctt ggccagttgc atgtcttctt tggaaaaatg 4680
tctattcagt tcctttgccc attttttaat tgggattttt ggtttcttgc tattgagttg 4740
tttgaattcg ctcgctttct tgctgtccaa tttctattaa aggttccttt gttccctaag 4800
tccaactact aaactggggg atattatgaa gggccttgag catctggatt ctgcctaata 4860
aaaaacattt attttcattg caagctcgct ttcttgctgt ccaatttcta ttaaaggttc 4920
ctttgttccc taagtccaac tactaaactg ggggatatta tgaagggcct tgagcatctg 4980
gattctgcct aataaaaaac atttattttc attgcaaaaa aaaaaaaaaa aaaaaaaaaa 5040
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5100
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaactc ggaaggacat ggtgtggaaa 5160
gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 5220
caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 5280
ttagtcagca accaaattaa agtaacccat aacttcgtat agcatacatt atacgaagtt 5340
atgaagttcc tattctctag aaagtatagg aacttctagt cacctatttc agcatactac 5400
gcgcgtagta tgctgaaata ggtttatcag cacacaatag tccattatac gcgcgtataa 5460
tggcaattgt gtgctgattg ggttacttta atttggatcc gtcgaccgat gcccttgaga 5520
gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 5580
atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc 5640
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 5700
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5760
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 5820
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 5880
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5940
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 6000
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 6060
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 6120
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 6180
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 6240
ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 6300
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 6360
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 6420
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 6480
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 6540
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 6600
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 6660
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 6720
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6780
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6840
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 6900
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 6960
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 7020
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 7080
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 7140
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 7200
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 7260
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 7320
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 7380
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 7440
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 7500
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 7560
tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 7620
cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 7680
cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 7740
tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 7800
gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 7860
tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 7920
ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 7980
taacgcgaat tttaacaaaa tattaacgct tacaatttgc cattcgccat tcaggctgcg 8040
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc 8100
atgataagta agtaatatta aggtacgtgg aggttttact tgctttaaaa aacctcccac 8160
acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 8220
cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 8280
tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat ggtactgtaa 8340
ctgagctaac ataa 8354
<210> 30
<211> 7710
<212> DNA
<213> artificial sequence
<220>
<223> SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-3'UTR[2huBGpA-A120]-SS*
<400> 30
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgcac acgaccacaa ttccactgaa agcattttaa 420
tacggaactt gtcactccca gggagcctcc gctcagccgg cagttggttc atttcaatcc 480
ccacgacaac ccttcaaagt gcagggcaga cagcaggtgg ctctgcccag gcgcctggat 540
cacagcccgg cctgcagccc tcacctgggc gcggggagac cctgaggacg ctcctccagg 600
cggcgctggc cggggcctgc ggacacggac gggcgggctg agctccggga cccctccccg 660
cgccccgcac cccgcacccc gcaccccgca ccccgcaccc ggcgctcacc cgtcccagcc 720
ccgccgcccg cagccccagc tgcaacgcag ccaccgccgc catcgcaccc ggccccgcgg 780
gcgcttccgg gacgcaggag gcatctgcat ccggggcgcc gctgagtccc gcccagagcc 840
ccgcccccgg ctccaggttc tgcgagcggc ttccgccggg ctgctccgcg ggcgcgtcgg 900
ccatgagcga gttgccgggc gacgtgcggg cgtttctgcg ggagcacccg agcctgcggc 960
tccagacgga cgcccgcaag gttcgcagcg cgggagggga acggagtggc ggagaagggc 1020
gcagttggga tgaggggctg aggggagggc aggggagagg agagggcagg ggagagggga 1080
gaggggagag caggagagag gggaaggcag gggagagggc gcggcgggat caggggagga 1140
gagggaaggg actttccggg gcggggcacg tggtgcacgg gactttccgt gcacgtgcac 1200
gggactttcc gggactttcc gggactttcc gtgcaccacg tggggacttt ccgtgcacga 1260
cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 1320
tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 1380
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 1440
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1500
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1560
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1620
gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 1680
tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 1740
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 1800
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctgccttc tccctcctgt 1860
gagtttggta agtcgacggg ccgggcctgg gccgggtccg ggccgggtcg ttggatcccc 1920
actacagccc gatactcaag cttgacgaat tcgagtatcc aaggtagtgg actagtgtga 1980
cgctgctgac ccctttcttt cccttctgca ggttggtgta cagtagcttc caaattgatt 2040
aattcgagcg aacgcgtcgc caccatgaac tccttctcca caagcgcctt cggtccagtt 2100
gccttctccc tgggcctgct cctggtgttg cctgctgcct tccctgcccc agtcttcaca 2160
ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt 2220
gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa 2280
aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat 2340
gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct 2400
gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt 2460
acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc 2520
aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg 2580
atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg 2640
ctgtgcgaac gcattctggc ggctagcgct actaacttca gcctgctgaa gcaggctgga 2700
gacgtggagg agaaccctgg acctggaagc ggagagggca gaggaagtct gctaacatgc 2760
ggtgacgtcg aggagaatcc tggacctgga tccggaatgg tgagcaaggg cgaggagctg 2820
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2880
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 2940
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 3000
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 3060
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 3120
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 3180
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 3240
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 3300
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 3360
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 3420
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3480
gggatcactc tcggcatgga cgagctgtac aagtaaaatc aacctctgga ttacaaaatt 3540
tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 3600
gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 3660
tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 3720
gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 3780
cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 3840
gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 3900
ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg 3960
cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 4020
ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 4080
atctcccttt gggccgcctc cccgcgctcg ctttcttgct gtccaatttc tattaaaggt 4140
tcctttgttc cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc 4200
tggattctgc ctaataaaaa acatttattt tcattgcaag ctcgctttct tgctgtccaa 4260
tttctattaa aggttccttt gttccctaag tccaactact aaactggggg atattatgaa 4320
gggccttgag catctggatt ctgcctaata aaaaacattt attttcattg caaaaaaaaa 4380
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactcggaa 4500
ggacatggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 4560
tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 4620
aaagcatgca tctcaattag tcagcaacca aattaaagta acccataact tcgtatagca 4680
tacattatac gaagttatga agttcctatt ctctagaaag tataggaact tctagtcacc 4740
tatttcagca tactacgcgc gtagtatgct gaaataggtt tatcagcaca caatagtcca 4800
ttatacgcgc gtataatggc aattgtgtgc tgattgggtt actttaattt ggatccgtcg 4860
accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac 4920
tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 4980
agcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 5040
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 5100
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 5160
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 5220
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 5280
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 5340
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 5400
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5460
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5520
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5580
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5640
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5700
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5760
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5820
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5880
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5940
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 6000
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 6060
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 6120
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 6180
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 6240
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 6300
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 6360
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 6420
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 6480
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 6540
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 6600
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 6660
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 6720
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 6780
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 6840
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 6900
cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 6960
ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 7020
tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 7080
ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 7140
atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 7200
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 7260
tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 7320
tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttgccatt 7380
cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac 7440
gccagcccaa gctaccatga taagtaagta atattaaggt acgtggaggt tttacttgct 7500
ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt 7560
gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 7620
acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta 7680
tcttatggta ctgtaactga gctaacataa 7710
<210> 31
<211> 8182
<212> DNA
<213> artificial sequence
<220>
<223> SS*-E1-CMV-UTR1-SecNLuc-2A-eGFP-MAR-WPRE-3'UTR[2huBGpA-A120]-SS*
<400> 31
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacggga ctttccgggg cggggcacgt ggtgcacggg 420
actttccgtg cacgtgcacg ggactttccg ggactttccg ggactttccg tgcaccacgt 480
ggggactttc cgtgcacgac attgattatt gactagttat taatagtaat caattacggg 540
gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 600
gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 660
agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 720
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 780
cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 840
gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 900
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 960
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 1020
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 1080
tctgccttct ccctcctgtg agtttggtaa gtcgacgggc cgggcctggg ccgggtccgg 1140
gccgggtcgt tggatcccca ctacagcccg atactcaagc ttgacgaatt cgagtatcca 1200
aggtagtgga ctagtgtgac gctgctgacc cctttctttc ccttctgcag gttggtgtac 1260
agtagcttcc aaattgatta attcgagcga acgcgtcgcc accatgaact ccttctccac 1320
aagcgccttc ggtccagttg ccttctccct gggcctgctc ctggtgttgc ctgctgcctt 1380
ccctgcccca gtcttcacac tcgaagattt cgttggggac tggcgacaga cagccggcta 1440
caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga atctcggggt 1500
gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga agatcgacat 1560
ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat 1620
ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc actatggcac 1680
actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc cgtatgaagg 1740
catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga acggcaacaa 1800
aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag taaccatcaa 1860
cggagtgacc ggctggcggc tgtgcgaacg cattctggcg gctagcgcta ctaacttcag 1920
cctgctgaag caggctggag acgtggagga gaaccctgga cctggaagcg gagagggcag 1980
aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat ccggaatggt 2040
gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 2100
cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa 2160
gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 2220
gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 2280
cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 2340
ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 2400
ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 2460
ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 2520
caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 2580
ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 2640
gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 2700
ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaaggatc 2760
ccattctcct tgatgtacta atttttcttt aaaagtgata ataatagctc ccatttagaa 2820
tttttaaata acacaacaaa tgtaaagtaa ctaatgtgtc ctctggatca tggtaagtaa 2880
tgaataaatt taactccctt taccttctcc ctttgctatt ttttccatgc taggatttat 2940
acatttttaa aaaactaaat ctgctatcaa atgacagctt taaatttact ttttaaaatt 3000
tgttattgta tatatttatg gggtataaag tgatgttatg atatatatat acacaatgta 3060
cactgattaa atcaagccaa ttaacatttt atcatctcaa atacttaaca ttttttgtag 3120
tgagaacatt tgaaatttac ttttagcaat ttcaaaacat acattattat tattaactat 3180
agtcaccatg atgtaccata gatctttaaa aacttattct tcctgcctaa ctgaaacttt 3240
gtactctttg actaacatct tttcattccc ccacttccca gcctctggta atcaccatta 3300
cacactctgc ttctatgagt tcaattgctt tagactccac gtaataaatg agatcatgca 3360
gcatttggct ttctgtgcct ggcttatcct tgcttagcat ggtgtcttac aggttcatcc 3420
atgttgcaac aaataacaga atctcattct ttgttaaggc tgaatactat tccattgggt 3480
atatatacca cattttcctt atccattaat ccactgatgg acccttaggt tgttgattcc 3540
atatattggc tattgtaaat agtgcagcaa tgaacatgag agtgcaacta tctcttcaat 3600
gtactgattt cgaatccttc ggatctatct cagaagtgag attgcaggat catataattc 3660
tacttttagt cttttgagga gctccataca gctttccata tggccatact aattacattc 3720
tcatcaacag tgtacaatgg tttccttttc tccacatcct caccaacatt tataattttt 3780
tgtctttttg ataatagcca tctgacaggt gtaaagtgat agctcattgc agttttaatt 3840
tgcatttttt gatgattagt aatgttgaga attttttcat atatctcttg gccagttgca 3900
tgtcttcttt ggaaaaatgt ctattcagtt cctttgccca ttttttaatt gggatttttg 3960
gtttcttgct attgagttgt ttgaattcaa tcaacctctg gattacaaaa tttgtgaaag 4020
attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 4080
gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 4140
ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 4200
cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 4260
ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 4320
tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 4380
gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 4440
gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 4500
gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 4560
ttgggccgcc tccccgcgct cgctttcttg ctgtccaatt tctattaaag gttcctttgt 4620
tccctaagtc caactactaa actgggggat attatgaagg gccttgagca tctggattct 4680
gcctaataaa aaacatttat tttcattgca agctcgcttt cttgctgtcc aatttctatt 4740
aaaggttcct ttgttcccta agtccaacta ctaaactggg ggatattatg aagggccttg 4800
agcatctgga ttctgcctaa taaaaaacat ttattttcat tgcaaaaaaa aaaaaaaaaa 4860
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4920
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaactcgg aaggacatgg 4980
tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 5040
tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 5100
catctcaatt agtcagcaac caaattaaag taacccataa cttcgtatag catacattat 5160
acgaagttat gaagttccta ttctctagaa agtataggaa cttctagtca cctatttcag 5220
catactacgc gcgtagtatg ctgaaatagg tttatcagca cacaatagtc cattatacgc 5280
gcgtataatg gcaattgtgt gctgattggg ttactttaat ttggatccgt cgaccgatgc 5340
ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg 5400
ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 5460
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 5520
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 5580
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 5640
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 5700
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 5760
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 5820
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 5880
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 5940
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 6000
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 6060
aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 6120
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 6180
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 6240
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 6300
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 6360
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 6420
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 6480
tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 6540
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 6600
cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 6660
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 6720
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 6780
aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 6840
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 6900
aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 6960
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 7020
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 7080
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 7140
gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 7200
ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 7260
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 7320
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 7380
gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc 7440
agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 7500
tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 7560
ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 7620
cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 7680
tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 7740
tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 7800
caaaaattta acgcgaattt taacaaaata ttaacgctta caatttgcca ttcgccattc 7860
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagccc 7920
aagctaccat gataagtaag taatattaag gtacgtggag gttttacttg ctttaaaaaa 7980
cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt 8040
gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 8100
agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatgg 8160
tactgtaact gagctaacat aa 8182
<210> 32
<211> 8943
<212> DNA
<213> artificial sequence
<220>
<223> SS*-UCOE-E1-CMV-UTR1-SecNLuc-2A-eGFP-MAR-WPRE-3'UTR[2huBGpA-A120]
-SS*
<400> 32
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgcac acgaccacaa ttccactgaa agcattttaa 420
tacggaactt gtcactccca gggagcctcc gctcagccgg cagttggttc atttcaatcc 480
ccacgacaac ccttcaaagt gcagggcaga cagcaggtgg ctctgcccag gcgcctggat 540
cacagcccgg cctgcagccc tcacctgggc gcggggagac cctgaggacg ctcctccagg 600
cggcgctggc cggggcctgc ggacacggac gggcgggctg agctccggga cccctccccg 660
cgccccgcac cccgcacccc gcaccccgca ccccgcaccc ggcgctcacc cgtcccagcc 720
ccgccgcccg cagccccagc tgcaacgcag ccaccgccgc catcgcaccc ggccccgcgg 780
gcgcttccgg gacgcaggag gcatctgcat ccggggcgcc gctgagtccc gcccagagcc 840
ccgcccccgg ctccaggttc tgcgagcggc ttccgccggg ctgctccgcg ggcgcgtcgg 900
ccatgagcga gttgccgggc gacgtgcggg cgtttctgcg ggagcacccg agcctgcggc 960
tccagacgga cgcccgcaag gttcgcagcg cgggagggga acggagtggc ggagaagggc 1020
gcagttggga tgaggggctg aggggagggc aggggagagg agagggcagg ggagagggga 1080
gaggggagag caggagagag gggaaggcag gggagagggc gcggcgggat caggggagga 1140
gagggaaggg actttccggg gcggggcacg tggtgcacgg gactttccgt gcacgtgcac 1200
gggactttcc gggactttcc gggactttcc gtgcaccacg tggggacttt ccgtgcacga 1260
cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca 1320
tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 1380
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 1440
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 1500
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 1560
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 1620
gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 1680
tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 1740
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 1800
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctgccttc tccctcctgt 1860
gagtttggta agtcgacggg ccgggcctgg gccgggtccg ggccgggtcg ttggatcccc 1920
actacagccc gatactcaag cttgacgaat tcgagtatcc aaggtagtgg actagtgtga 1980
cgctgctgac ccctttcttt cccttctgca ggttggtgta cagtagcttc caaattgatt 2040
aattcgagcg aacgcgtcgc caccatgaac tccttctcca caagcgcctt cggtccagtt 2100
gccttctccc tgggcctgct cctggtgttg cctgctgcct tccctgcccc agtcttcaca 2160
ctcgaagatt tcgttgggga ctggcgacag acagccggct acaacctgga ccaagtcctt 2220
gaacagggag gtgtgtccag tttgtttcag aatctcgggg tgtccgtaac tccgatccaa 2280
aggattgtcc tgagcggtga aaatgggctg aagatcgaca tccatgtcat catcccgtat 2340
gaaggtctga gcggcgacca aatgggccag atcgaaaaaa tttttaaggt ggtgtaccct 2400
gtggatgatc atcactttaa ggtgatcctg cactatggca cactggtaat cgacggggtt 2460
acgccgaaca tgatcgacta tttcggacgg ccgtatgaag gcatcgccgt gttcgacggc 2520
aaaaagatca ctgtaacagg gaccctgtgg aacggcaaca aaattatcga cgagcgcctg 2580
atcaaccccg acggctccct gctgttccga gtaaccatca acggagtgac cggctggcgg 2640
ctgtgcgaac gcattctggc ggctagcgct actaacttca gcctgctgaa gcaggctgga 2700
gacgtggagg agaaccctgg acctggaagc ggagagggca gaggaagtct gctaacatgc 2760
ggtgacgtcg aggagaatcc tggacctgga tccggaatgg tgagcaaggg cgaggagctg 2820
ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2880
agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 2940
tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 3000
gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 3060
atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 3120
acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 3180
atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 3240
cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 3300
cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 3360
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 3420
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3480
gggatcactc tcggcatgga cgagctgtac aagtaaggat cccattctcc ttgatgtact 3540
aatttttctt taaaagtgat aataatagct cccatttaga atttttaaat aacacaacaa 3600
atgtaaagta actaatgtgt cctctggatc atggtaagta atgaataaat ttaactccct 3660
ttaccttctc cctttgctat tttttccatg ctaggattta tacattttta aaaaactaaa 3720
tctgctatca aatgacagct ttaaatttac tttttaaaat ttgttattgt atatatttat 3780
ggggtataaa gtgatgttat gatatatata tacacaatgt acactgatta aatcaagcca 3840
attaacattt tatcatctca aatacttaac attttttgta gtgagaacat ttgaaattta 3900
cttttagcaa tttcaaaaca tacattatta ttattaacta tagtcaccat gatgtaccat 3960
agatctttaa aaacttattc ttcctgccta actgaaactt tgtactcttt gactaacatc 4020
ttttcattcc cccacttccc agcctctggt aatcaccatt acacactctg cttctatgag 4080
ttcaattgct ttagactcca cgtaataaat gagatcatgc agcatttggc tttctgtgcc 4140
tggcttatcc ttgcttagca tggtgtctta caggttcatc catgttgcaa caaataacag 4200
aatctcattc tttgttaagg ctgaatacta ttccattggg tatatatacc acattttcct 4260
tatccattaa tccactgatg gacccttagg ttgttgattc catatattgg ctattgtaaa 4320
tagtgcagca atgaacatga gagtgcaact atctcttcaa tgtactgatt tcgaatcctt 4380
cggatctatc tcagaagtga gattgcagga tcatataatt ctacttttag tcttttgagg 4440
agctccatac agctttccat atggccatac taattacatt ctcatcaaca gtgtacaatg 4500
gtttcctttt ctccacatcc tcaccaacat ttataatttt ttgtcttttt gataatagcc 4560
atctgacagg tgtaaagtga tagctcattg cagttttaat ttgcattttt tgatgattag 4620
taatgttgag aattttttca tatatctctt ggccagttgc atgtcttctt tggaaaaatg 4680
tctattcagt tcctttgccc attttttaat tgggattttt ggtttcttgc tattgagttg 4740
tttgaattca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac 4800
tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt 4860
gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat 4920
gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca 4980
acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc 5040
cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg 5100
gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct 5160
tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct 5220
tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt 5280
ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgc 5340
tcgctttctt gctgtccaat ttctattaaa ggttcctttg ttccctaagt ccaactacta 5400
aactggggga tattatgaag ggccttgagc atctggattc tgcctaataa aaaacattta 5460
ttttcattgc aagctcgctt tcttgctgtc caatttctat taaaggttcc tttgttccct 5520
aagtccaact actaaactgg gggatattat gaagggcctt gagcatctgg attctgccta 5580
ataaaaaaca tttattttca ttgcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5640
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5700
aaaaaaaaaa aaaaaaaaaa aaaaaactcg gaaggacatg gtgtggaaag tccccaggct 5760
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 5820
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 5880
ccaaattaaa gtaacccata acttcgtata gcatacatta tacgaagtta tgaagttcct 5940
attctctaga aagtatagga acttctagtc acctatttca gcatactacg cgcgtagtat 6000
gctgaaatag gtttatcagc acacaatagt ccattatacg cgcgtataat ggcaattgtg 6060
tgctgattgg gttactttaa tttggatccg tcgaccgatg cccttgagag ccttcaaccc 6120
agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt 6180
ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg 6240
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6300
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6360
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6420
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6480
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6540
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6600
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 6660
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6720
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6780
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6840
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6900
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 6960
agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 7020
acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 7080
tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 7140
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 7200
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 7260
agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 7320
cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 7380
ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 7440
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 7500
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 7560
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 7620
tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 7680
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 7740
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 7800
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 7860
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 7920
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 7980
aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 8040
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 8100
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct 8160
gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 8220
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 8280
gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 8340
ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 8400
gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 8460
tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 8520
tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 8580
ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg 8640
aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa 8700
gtaatattaa ggtacgtgga ggttttactt gctttaaaaa acctcccaca cctccccctg 8760
aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat 8820
ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 8880
tctagttgtg gtttgtccaa actcatcaat gtatcttatg gtactgtaac tgagctaaca 8940
taa 8943
<210> 33
<211> 340
<212> DNA
<213> artificial sequence
<220>
<223> super sequence, SS
<400> 33
taaagtaacc caatcagcac acaattgcca ttatacgcgc gtataatgga ctattgtgtg 60
ctgataaacc tatttcagca tactacgcgc gtagtatgct gaaataggtg actagaagtt 120
cctatacttt ctagagaata ggaacttcat aacttcgtat aatgtatgct atacgaagtt 180
atgggttact ttaatttggt tgctgactaa ttgagatgca tgctttgcat acttctgcct 240
gctggggagc ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc 300
atacttctgc ctgctgggga gcctggggac tttccacacc 340
<210> 34
<211> 6326
<212> DNA
<213> artificial sequence
<220>
<223> pGL2-CAG-SecNLuc-2A-eGFP-WPRE-bGlobin polyA
<400> 34
cccgggaggt accgagctct tacgcgtgct agcctgggtc gacattgatt attgactagt 60
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 120
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 180
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 240
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 300
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 360
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 420
gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca 480
attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg 540
gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggaaaggtgc 600
ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 660
gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgt tgccttcgcc 720
ccgtgccccg ctccgcgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact 780
cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta 840
atgacggctc gtttcttttc tgtggctgcg tgaaagcctt aaagggctcc gggagggccc 900
tttgtgcggg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg 960
cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg 1020
cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg 1080
gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg 1140
tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc ctccccgagt tgctgagcac 1200
ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc 1260
ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc 1320
tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc 1380
agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc 1440
tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag 1500
cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc 1560
cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg 1620
ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct 1680
aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg 1740
ctgtctcatc attttggcaa agaattgatt aattcgagcg aacgcgtcgc caccatgaac 1800
tccttctcca caagcgcctt cggtccagtt gccttctccc tgggcctgct cctggtgttg 1860
cctgctgcct tccctgcccc agtcttcaca ctcgaagatt tcgttgggga ctggcgacag 1920
acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag 1980
aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg 2040
aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag 2100
atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg 2160
cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg 2220
ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg 2280
aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga 2340
gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc ggctagcgct 2400
actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg acctggaagc 2460
ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggacctgga 2520
tccggaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 2580
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 2640
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 2700
cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 2760
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 2820
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 2880
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 2940
gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 3000
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 3060
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 3120
aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 3180
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 3240
aagtaagcgg ccgcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg 3300
ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg 3360
acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg 3420
caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacattggat ccgtcgaccg 3480
atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 3540
gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 3600
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 3660
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 3720
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 3780
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 3840
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 3900
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 3960
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4020
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4080
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 4140
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 4200
gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 4260
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 4320
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 4380
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4440
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 4500
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 4560
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 4620
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 4680
gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 4740
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 4800
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 4860
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 4920
atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 4980
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 5040
gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 5100
aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 5160
acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 5220
ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 5280
tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 5340
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 5400
catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 5460
atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 5520
aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 5580
gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 5640
ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 5700
agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 5760
ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 5820
gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 5880
ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 5940
ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt gccattcgcc 6000
attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 6060
gcccaagcta ccatgataag taagtaatat taaggtacgt ggaggtttta cttgctttaa 6120
aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta 6180
acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 6240
ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 6300
atggtactgt aactgagcta acataa 6326
<210> 35
<211> 1626
<212> DNA
<213> artificial sequence
<220>
<223> CAG [ E1X3+CBA promoter+intron ]
<400> 35
gggactttcc ggggcggggc acgtggtgca cgggactttc cgtgcacgtg cacgggactt 60
tccgggactt tccgggactt tccgtgcacc acgtggggac tttccgtgca cgggactttc 120
cggggcgggg cacgtggtgc acgggacttt ccgtgcacgt gcacgggact ttccgggact 180
ttccgggact ttccgtgcac cacgtgggga ctttccgtgc acgggacttt ccggggcggg 240
gcacgtggtg cacgggactt tccgtgcacg tgcacgggac tttccgggac tttccgggac 300
tttccgtgca ccacgtgggg actttccgtg cacgtcgagg tgagccccac gttctgcttc 360
actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 420
ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 480
cgaggggcgg ggcggggcga ggcggaaagg tgcggcggca gccaatcaga gcggcgcgct 540
ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 600
gcggcgggcg ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg 660
ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc 720
ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct 780
gcgtgaaagc cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg 840
gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct 900
gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg 960
cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc 1020
ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc 1080
cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt 1140
acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg 1200
ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg 1260
agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc 1320
gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc 1380
cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg 1440
ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg 1500
ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc 1560
tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc 1620
ctacag 1626
<210> 36
<211> 1559
<212> DNA
<213> artificial sequence
<220>
<223> CAG [ E2+CBA promoter+intron ]
<400> 36
tgggactttc cactagacat gacacagcaa tctgatatgc ttgcgtgaga agaggattca 60
tatcctggga ctttccacag attttaccgg aagttgttag atgcttgcgt gagaagatct 120
aacatgacac agcaatcctt agtgggactt tccaagtatg tggggcgggg agtatacatg 180
acacagcaat tgatcattac cggaagttta taggtgggac tttccagacc tatgcttgcg 240
tgagaagaaa ggtctgggac tttccagtcg aggtgagccc cacgttctgc ttcactctcc 300
ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg 360
cagcgatggg ggcggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg 420
cggggcgggg cgaggcggaa aggtgcggcg gcagccaatc agagcggcgc gctccgaaag 480
tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg 540
gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gcgccgcctc gcgccgcccg 600
ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct 660
ccgggctgta attagcgctt ggtttaatga cggctcgttt cttttctgtg gctgcgtgaa 720
agccttaaag ggctccggga gggccctttg tgcggggggg agcggctcgg ggggtgcgtg 780
cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 840
ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 900
gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 960
gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc gggctgtaac ccccccctgc 1020
acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc 1080
gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg 1140
cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg 1200
gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg 1260
cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca 1320
ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg 1380
agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc 1440
cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg 1500
tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacag 1559
<210> 37
<211> 826
<212> DNA
<213> artificial sequence
<220>
<223> CAG [ E1X3+CBA promoter+UTR1 ]
<400> 37
gggactttcc ggggcggggc acgtggtgca cgggactttc cgtgcacgtg cacgggactt 60
tccgggactt tccgggactt tccgtgcacc acgtggggac tttccgtgca cgggactttc 120
cggggcgggg cacgtggtgc acgggacttt ccgtgcacgt gcacgggact ttccgggact 180
ttccgggact ttccgtgcac cacgtgggga ctttccgtgc acgggacttt ccggggcggg 240
gcacgtggtg cacgggactt tccgtgcacg tgcacgggac tttccgggac tttccgggac 300
tttccgtgca ccacgtgggg actttccgtg cacgtcgagg tgagccccac gttctgcttc 360
actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta 420
ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 480
cgaggggcgg ggcggggcga ggcggaaagg tgcggcggca gccaatcaga gcggcgcgct 540
ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 600
gcggcgggcg ctgccttctc cctcctgtga gtttggtaag tcgacgggcc gggcctgggc 660
cgggtccggg ccgggtcgtt ggatccccac tacagcccga tactcaagct tgacgaattc 720
gagtatccaa ggtagtggac tagtgtgacg ctgctgaccc ctttctttcc cttctgcagg 780
ttggtgtaca gtagcttcca aattgattaa ttcgagcgaa cgcgtc 826
<210> 38
<211> 759
<212> DNA
<213> artificial sequence
<220>
<223> CAG [ E2 (U100) +CBA promoter+UTR1 ]
<400> 38
tgggactttc cactagacat gacacagcaa tctgatatgc ttgcgtgaga agaggattca 60
tatcctggga ctttccacag attttaccgg aagttgttag atgcttgcgt gagaagatct 120
aacatgacac agcaatcctt agtgggactt tccaagtatg tggggcgggg agtatacatg 180
acacagcaat tgatcattac cggaagttta taggtgggac tttccagacc tatgcttgcg 240
tgagaagaaa ggtctgggac tttccagtcg aggtgagccc cacgttctgc ttcactctcc 300
ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg 360
cagcgatggg ggcggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg 420
cggggcgggg cgaggcggaa aggtgcggcg gcagccaatc agagcggcgc gctccgaaag 480
tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg 540
gcgctgcctt ctccctcctg tgagtttggt aagtcgacgg gccgggcctg ggccgggtcc 600
gggccgggtc gttggatccc cactacagcc cgatactcaa gcttgacgaa ttcgagtatc 660
caaggtagtg gactagtgtg acgctgctga cccctttctt tcccttctgc aggttggtgt 720
acagtagctt ccaaattgat taattcgagc gaacgcgtc 759
<210> 39
<211> 808
<212> DNA
<213> artificial sequence
<220>
<223> CMV enhancer-EF 1-UTR1
<400> 39
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gggcagagcg cacatcgccc acagtccccg agaagttggg 420
gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 480
tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 540
agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agctgccttc 600
tccctcctgt gagtttggta agtcgacggg ccgggcctgg gccgggtccg ggccgggtcg 660
ttggatcccc actacagccc gatactcaag cttgacgaat tcgagtatcc aaggtagtgg 720
actagtgtga cgctgctgac ccctttcttt cccttctgca ggttggtgta cagtagcttc 780
caaattgatt aattcgagcg aacgcgtc 808
<210> 40
<211> 7772
<212> DNA
<213> artificial sequence
<220>
<223> 40-1 pGL2-SS-CAG [ CMV enhancer+CBA
Promoter+intron ] -SecNLuc-2A-eGFP-WPRE-3' utr (108 to 120 polyA) -SS
<400> 40
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgaca ttgattattg actagttatt aatagtaatc 420
aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 480
aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 540
tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 600
gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 660
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 720
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc 780
cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt 840
tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggcgc gcgccaggcg 900
gggcggggcg gggcgagggg cggggcgggg cgaggcggaa aggtgcggcg gcagccaatc 960
agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 1020
aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc 1080
gcgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca caggtgagcg 1140
ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga cggctcgttt 1200
cttttctgtg gctgcgtgaa agccttaaag ggctccggga gggccctttg tgcggggggg 1260
agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg 1320
ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg 1380
cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac 1440
aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcggcggtc 1500
gggctgtaac ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 1560
tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 1620
ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 1680
cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 1740
atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 1800
atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 1860
caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 1920
tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1980
ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 2040
cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 2100
tggcaaagaa ttgattaatt cgagcgaacg cgtcgccacc atgaactcct tctccacaag 2160
cgccttcggt ccagttgcct tctccctggg cctgctcctg gtgttgcctg ctgccttccc 2220
tgccccagtc ttcacactcg aagatttcgt tggggactgg cgacagacag ccggctacaa 2280
cctggaccaa gtccttgaac agggaggtgt gtccagtttg tttcagaatc tcggggtgtc 2340
cgtaactccg atccaaagga ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca 2400
tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt 2460
taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgcact atggcacact 2520
ggtaatcgac ggggttacgc cgaacatgat cgactatttc ggacggccgt atgaaggcat 2580
cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg gcaacaaaat 2640
tatcgacgag cgcctgatca accccgacgg ctccctgctg ttccgagtaa ccatcaacgg 2700
agtgaccggc tggcggctgt gcgaacgcat tctggcggct agcgctacta acttcagcct 2760
gctgaagcag gctggagacg tggaggagaa ccctggacct ggaagcggag agggcagagg 2820
aagtctgcta acatgcggtg acgtcgagga gaatcctgga cctggatccg gaatggtgag 2880
caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 2940
aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 3000
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 3060
caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 3120
cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 3180
cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 3240
catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 3300
gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 3360
ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 3420
ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 3480
cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 3540
gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaaatcaacc 3600
tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 3660
gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 3720
cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 3780
tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 3840
cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 3900
ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3960
tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 4020
tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 4080
ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 4140
ccctcagacg agtcggatct ccctttgggc cgcctccccg cgctcgcttt cttgctgtcc 4200
aatttctatt aaaggttcct ttgttcccta agtccaacta ctaaactggg ggatattatg 4260
aagggccttg agcatctgga ttctgcctaa taaaaaacat ttattttcat tgcaagctcg 4320
ctttcttgct gtccaatttc tattaaaggt tcctttgttc cctaagtcca actactaaac 4380
tgggggatat tatgaagggc cttgagcatc tggattctgc ctaataaaaa acatttattt 4440
tcattgcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaactcgg 4560
aaggacatgg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 4620
tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 4680
gcaaagcatg catctcaatt agtcagcaac caaattaaag taacccataa cttcgtatag 4740
catacattat acgaagttat gaagttccta ttctctagaa agtataggaa cttctagtca 4800
cctatttcag catactacgc gcgtagtatg ctgaaatagg tttatcagca cacaatagtc 4860
cattatacgc gcgtataatg gcaattgtgt gctgattggg ttactttaat ttggatccgt 4920
cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg 4980
actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg 5040
gcagcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 5100
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 5160
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 5220
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 5280
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 5340
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 5400
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 5460
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 5520
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 5580
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5640
gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 5700
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 5760
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 5820
agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5880
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 5940
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 6000
aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 6060
ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 6120
gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 6180
aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 6240
ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6300
tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 6360
ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6420
cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6480
agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 6540
gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6600
gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 6660
acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 6720
acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 6780
agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 6840
aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6900
gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6960
tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 7020
ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 7080
cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 7140
ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 7200
tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 7260
gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 7320
ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 7380
gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttgcca 7440
ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 7500
acgccagccc aagctaccat gataagtaag taatattaag gtacgtggag gttttacttg 7560
ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg 7620
ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 7680
tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 7740
tatcttatgg tactgtaact gagctaacat aa 7772
<210> 41
<211> 7725
<212> DNA
<213> artificial sequence
<220>
<223> 4-2 pGL2-SS*-CAG [E1 X3+CBA
Promoter+intron e ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120)
polyA)-SS*
<400> 41
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacggga ctttccgggg cggggcacgt ggtgcacggg 420
actttccgtg cacgtgcacg ggactttccg ggactttccg ggactttccg tgcaccacgt 480
ggggactttc cgtgcacggg actttccggg gcggggcacg tggtgcacgg gactttccgt 540
gcacgtgcac gggactttcc gggactttcc gggactttcc gtgcaccacg tggggacttt 600
ccgtgcacgg gactttccgg ggcggggcac gtggtgcacg ggactttccg tgcacgtgca 660
cgggactttc cgggactttc cgggactttc cgtgcaccac gtggggactt tccgtgcacg 720
tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 780
ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 840
cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gaaaggtgcg 900
gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg 960
cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgtt gccttcgccc 1020
cgtgccccgc tccgcgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 1080
ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa 1140
tgacggctcg tttcttttct gtggctgcgt gaaagcctta aagggctccg ggagggccct 1200
ttgtgcgggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc 1260
gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc 1320
gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg 1380
ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt 1440
gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt gctgagcacg 1500
gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg 1560
gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct 1620
cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca 1680
gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct 1740
gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc 1800
ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc 1860
gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg 1920
gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta 1980
accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc 2040
tgtctcatca ttttggcaaa gaattgatta attcgagcga acgcgtcgcc accatgaact 2100
ccttctccac aagcgccttc ggtccagttg ccttctccct gggcctgctc ctggtgttgc 2160
ctgctgcctt ccctgcccca gtcttcacac tcgaagattt cgttggggac tggcgacaga 2220
cagccggcta caacctggac caagtccttg aacagggagg tgtgtccagt ttgtttcaga 2280
atctcggggt gtccgtaact ccgatccaaa ggattgtcct gagcggtgaa aatgggctga 2340
agatcgacat ccatgtcatc atcccgtatg aaggtctgag cggcgaccaa atgggccaga 2400
tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca tcactttaag gtgatcctgc 2460
actatggcac actggtaatc gacggggtta cgccgaacat gatcgactat ttcggacggc 2520
cgtatgaagg catcgccgtg ttcgacggca aaaagatcac tgtaacaggg accctgtgga 2580
acggcaacaa aattatcgac gagcgcctga tcaaccccga cggctccctg ctgttccgag 2640
taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg cattctggcg gctagcgcta 2700
ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctggaagcg 2760
gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat 2820
ccggaatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc 2880
tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca 2940
cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc 3000
ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca 3060
tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca 3120
tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca 3180
ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg 3240
ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga 3300
agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc 3360
tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca 3420
accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca 3480
tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca 3540
agtaaaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 3600
ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 3660
cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg 3720
agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 3780
ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 3840
tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 3900
ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc 3960
tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 4020
ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 4080
gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgctcgc 4140
tttcttgctg tccaatttct attaaaggtt cctttgttcc ctaagtccaa ctactaaact 4200
gggggatatt atgaagggcc ttgagcatct ggattctgcc taataaaaaa catttatttt 4260
cattgcaagc tcgctttctt gctgtccaat ttctattaaa ggttcctttg ttccctaagt 4320
ccaactacta aactggggga tattatgaag ggccttgagc atctggattc tgcctaataa 4380
aaaacattta ttttcattgc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4500
aaaaaaaact cggaaggaca tggtgtggaa agtccccagg ctccccagca ggcagaagta 4560
tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 4620
caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaaatta aagtaaccca 4680
taacttcgta tagcatacat tatacgaagt tatgaagttc ctattctcta gaaagtatag 4740
gaacttctag tcacctattt cagcatacta cgcgcgtagt atgctgaaat aggtttatca 4800
gcacacaata gtccattata cgcgcgtata atggcaattg tgtgctgatt gggttacttt 4860
aatttggatc cgtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg 4920
ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt 4980
aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 5040
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5100
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5160
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 5220
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 5280
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5340
ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 5400
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5460
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5520
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5580
cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 5640
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 5700
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 5760
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 5820
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 5880
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 5940
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6000
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 6060
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 6120
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 6180
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 6240
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 6300
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 6360
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 6420
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 6480
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 6540
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 6600
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 6660
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 6720
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 6780
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 6840
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 6900
ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg 6960
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 7020
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 7080
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 7140
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 7200
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 7260
tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 7320
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 7380
ttacaatttg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 7440
cctcttcgct attacgccag cccaagctac catgataagt aagtaatatt aaggtacgtg 7500
gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg 7560
aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat 7620
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 7680
aaactcatca atgtatctta tggtactgta actgagctaa cataa 7725
<210> 42
<211> 7658
<212> DNA
<213> artificial sequence
<220>
<223> 4-3 pGL2-SS*-CAG [E2(U100)+CBA
Promoter+intron e ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120)
polyA)-SS*
<400> 42
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgactggg actttccact agacatgaca cagcaatctg 420
atatgcttgc gtgagaagag gattcatatc ctgggacttt ccacagattt taccggaagt 480
tgttagatgc ttgcgtgaga agatctaaca tgacacagca atccttagtg ggactttcca 540
agtatgtggg gcggggagta tacatgacac agcaattgat cattaccgga agtttatagg 600
tgggactttc cagacctatg cttgcgtgag aagaaaggtc tgggactttc cagtcgaggt 660
gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta 720
tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg gggcgcgcgc 780
caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggaaaggt gcggcggcag 840
ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc 900
cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg ccccgtgccc 960
cgctccgcgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg 1020
tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc 1080
tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc cctttgtgcg 1140
ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc 1200
tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc 1260
agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag 1320
gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg 1380
gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc 1440
ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg 1500
gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga 1560
ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg 1620
ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga 1680
gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg 1740
cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct 1800
tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg 1860
cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt 1920
tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca 1980
tcattttggc aaagaattga ttaattcgag cgaacgcgtc gccaccatga actccttctc 2040
cacaagcgcc ttcggtccag ttgccttctc cctgggcctg ctcctggtgt tgcctgctgc 2100
cttccctgcc ccagtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg 2160
ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg 2220
ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga 2280
catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa 2340
aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg 2400
cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga 2460
aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa 2520
caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat 2580
caacggagtg accggctggc ggctgtgcga acgcattctg gcggctagcg ctactaactt 2640
cagcctgctg aagcaggctg gagacgtgga ggagaaccct ggacctggaa gcggagaggg 2700
cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggacctg gatccggaat 2760
ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 2820
cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 2880
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 2940
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 3000
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 3060
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 3120
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 3180
gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 3240
catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 3300
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 3360
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 3420
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaaa 3480
tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 3540
ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 3600
ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 3660
gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 3720
ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 3780
tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 3840
gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 3900
ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 3960
tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 4020
ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgct cgctttcttg 4080
ctgtccaatt tctattaaag gttcctttgt tccctaagtc caactactaa actgggggat 4140
attatgaagg gccttgagca tctggattct gcctaataaa aaacatttat tttcattgca 4200
agctcgcttt cttgctgtcc aatttctatt aaaggttcct ttgttcccta agtccaacta 4260
ctaaactggg ggatattatg aagggccttg agcatctgga ttctgcctaa taaaaaacat 4320
ttattttcat tgcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4380
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440
actcggaagg acatggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 4500
catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 4560
aagtatgcaa agcatgcatc tcaattagtc agcaaccaaa ttaaagtaac ccataacttc 4620
gtatagcata cattatacga agttatgaag ttcctattct ctagaaagta taggaacttc 4680
tagtcaccta tttcagcata ctacgcgcgt agtatgctga aataggttta tcagcacaca 4740
atagtccatt atacgcgcgt ataatggcaa ttgtgtgctg attgggttac tttaatttgg 4800
atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg 4860
ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag 4920
gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 4980
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 5040
taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 5100
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 5160
ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 5220
aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 5280
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 5340
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 5400
cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 5460
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 5520
cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 5580
gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 5640
cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 5700
tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 5760
ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 5820
aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 5880
atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 5940
ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 6000
tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 6060
agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 6120
taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 6180
tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 6240
cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 6300
ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 6360
tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 6420
tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 6480
cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 6540
tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 6600
gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 6660
tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 6720
atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 6780
tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 6840
cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg 6900
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 6960
cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 7020
ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 7080
ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 7140
gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 7200
tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 7260
aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat 7320
ttgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc 7380
gctattacgc cagcccaagc taccatgata agtaagtaat attaaggtac gtggaggttt 7440
tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa 7500
ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 7560
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 7620
tcaatgtatc ttatggtact gtaactgagc taacataa 7658
<210> 43
<211> 6850
<212> DNA
<213> artificial sequence
<220>
<223> 4-4 pGL2-SS*-CAG [E1 X3+CBA
promoter+UTR1 ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS
<400> 43
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacggga ctttccgggg cggggcacgt ggtgcacggg 420
actttccgtg cacgtgcacg ggactttccg ggactttccg ggactttccg tgcaccacgt 480
ggggactttc cgtgcacggg actttccggg gcggggcacg tggtgcacgg gactttccgt 540
gcacgtgcac gggactttcc gggactttcc gggactttcc gtgcaccacg tggggacttt 600
ccgtgcacgg gactttccgg ggcggggcac gtggtgcacg ggactttccg tgcacgtgca 660
cgggactttc cgggactttc cgggactttc cgtgcaccac gtggggactt tccgtgcacg 720
tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 780
ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 840
cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gaaaggtgcg 900
gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg 960
cggcggccct ataaaaagcg aagcgcgcgg cgggcgctgc cttctccctc ctgtgagttt 1020
ggtaagtcga cgggccgggc ctgggccggg tccgggccgg gtcgttggat ccccactaca 1080
gcccgatact caagcttgac gaattcgagt atccaaggta gtggactagt gtgacgctgc 1140
tgaccccttt ctttcccttc tgcaggttgg tgtacagtag cttccaaatt gattaattcg 1200
agcgaacgcg tcgccaccat gaactccttc tccacaagcg ccttcggtcc agttgccttc 1260
tccctgggcc tgctcctggt gttgcctgct gccttccctg ccccagtctt cacactcgaa 1320
gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag 1380
ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt 1440
gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt 1500
ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat 1560
gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg 1620
aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag 1680
atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac 1740
cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc 1800
gaacgcattc tggcggctag cgctactaac ttcagcctgc tgaagcaggc tggagacgtg 1860
gaggagaacc ctggacctgg aagcggagag ggcagaggaa gtctgctaac atgcggtgac 1920
gtcgaggaga atcctggacc tggatccgga atggtgagca agggcgagga gctgttcacc 1980
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 2040
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 2100
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag 2160
tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 2220
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 2280
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 2340
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 2400
gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 2460
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 2520
gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa 2580
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 2640
actctcggca tggacgagct gtacaagtaa aatcaacctc tggattacaa aatttgtgaa 2700
agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta 2760
atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa 2820
tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg 2880
tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc 2940
ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc 3000
cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg 3060
gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg 3120
acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg 3180
ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc 3240
ctttgggccg cctccccgcg ctcgctttct tgctgtccaa tttctattaa aggttccttt 3300
gttccctaag tccaactact aaactggggg atattatgaa gggccttgag catctggatt 3360
ctgcctaata aaaaacattt attttcattg caagctcgct ttcttgctgt ccaatttcta 3420
ttaaaggttc ctttgttccc taagtccaac tactaaactg ggggatatta tgaagggcct 3480
tgagcatctg gattctgcct aataaaaaac atttattttc attgcaaaaa aaaaaaaaaa 3540
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaactcggaa ggacatggtg tggaaagtcc 3660
ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg 3720
tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 3780
tcagcaacca aattaaagta acccataact tcgtatagca tacattatac gaagttatga 3840
agttcctatt ctctagaaag tataggaact tctagtcacc tatttcagca tactacgcgc 3900
gtagtatgct gaaataggtt tatcagcaca caatagtcca ttatacgcgc gtataatggc 3960
aattgtgtgc tgattgggtt actttaattt ggatccgtcg accgatgccc ttgagagcct 4020
tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga 4080
ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg 4140
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4200
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4260
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4320
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4380
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4440
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4500
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4560
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4620
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4680
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4740
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 4800
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 4860
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 4920
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 4980
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 5040
atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5100
gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5160
atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5220
ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5280
cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5340
agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5400
cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 5460
tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 5520
agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 5580
gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 5640
gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 5700
ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 5760
tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 5820
tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 5880
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 5940
caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 6000
atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 6060
gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 6120
acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 6180
ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 6240
gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 6300
tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 6360
ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 6420
gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 6480
gcgaatttta acaaaatatt aacgcttaca atttgccatt cgccattcag gctgcgcaac 6540
tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagcccaa gctaccatga 6600
taagtaagta atattaaggt acgtggaggt tttacttgct ttaaaaaacc tcccacacct 6660
ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc 6720
ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 6780
actgcattct agttgtggtt tgtccaaact catcaatgta tcttatggta ctgtaactga 6840
gctaacataa 6850
<210> 44
<211> 6783
<212> DNA
<213> artificial sequence
<220>
<223> 4-5-pGL2-SS*-CAG [E2 (U100)+CBA
promoter+UTR1 ] -SecNLuc-2A-eGFP-WPRE-3' UTR (108 to 120 polyA) -SS
<400> 44
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgactggg actttccact agacatgaca cagcaatctg 420
atatgcttgc gtgagaagag gattcatatc ctgggacttt ccacagattt taccggaagt 480
tgttagatgc ttgcgtgaga agatctaaca tgacacagca atccttagtg ggactttcca 540
agtatgtggg gcggggagta tacatgacac agcaattgat cattaccgga agtttatagg 600
tgggactttc cagacctatg cttgcgtgag aagaaaggtc tgggactttc cagtcgaggt 660
gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta 720
tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg gggcgcgcgc 780
caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggaaaggt gcggcggcag 840
ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc 900
cctataaaaa gcgaagcgcg cggcgggcgc tgccttctcc ctcctgtgag tttggtaagt 960
cgacgggccg ggcctgggcc gggtccgggc cgggtcgttg gatccccact acagcccgat 1020
actcaagctt gacgaattcg agtatccaag gtagtggact agtgtgacgc tgctgacccc 1080
tttctttccc ttctgcaggt tggtgtacag tagcttccaa attgattaat tcgagcgaac 1140
gcgtcgccac catgaactcc ttctccacaa gcgccttcgg tccagttgcc ttctccctgg 1200
gcctgctcct ggtgttgcct gctgccttcc ctgccccagt cttcacactc gaagatttcg 1260
ttggggactg gcgacagaca gccggctaca acctggacca agtccttgaa cagggaggtg 1320
tgtccagttt gtttcagaat ctcggggtgt ccgtaactcc gatccaaagg attgtcctga 1380
gcggtgaaaa tgggctgaag atcgacatcc atgtcatcat cccgtatgaa ggtctgagcg 1440
gcgaccaaat gggccagatc gaaaaaattt ttaaggtggt gtaccctgtg gatgatcatc 1500
actttaaggt gatcctgcac tatggcacac tggtaatcga cggggttacg ccgaacatga 1560
tcgactattt cggacggccg tatgaaggca tcgccgtgtt cgacggcaaa aagatcactg 1620
taacagggac cctgtggaac ggcaacaaaa ttatcgacga gcgcctgatc aaccccgacg 1680
gctccctgct gttccgagta accatcaacg gagtgaccgg ctggcggctg tgcgaacgca 1740
ttctggcggc tagcgctact aacttcagcc tgctgaagca ggctggagac gtggaggaga 1800
accctggacc tggaagcgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg 1860
agaatcctgg acctggatcc ggaatggtga gcaagggcga ggagctgttc accggggtgg 1920
tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 1980
agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 2040
agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 2100
gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 2160
acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 2220
tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 2280
aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 2340
tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 2400
aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 2460
ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 2520
acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 2580
gcatggacga gctgtacaag taaaatcaac ctctggatta caaaatttgt gaaagattga 2640
ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 2700
tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 2760
tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 2820
tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 2880
ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 2940
gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 3000
catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 3060
tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 3120
ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 3180
ccgcctcccc gcgctcgctt tcttgctgtc caatttctat taaaggttcc tttgttccct 3240
aagtccaact actaaactgg gggatattat gaagggcctt gagcatctgg attctgccta 3300
ataaaaaaca tttattttca ttgcaagctc gctttcttgc tgtccaattt ctattaaagg 3360
ttcctttgtt ccctaagtcc aactactaaa ctgggggata ttatgaaggg ccttgagcat 3420
ctggattctg cctaataaaa aacatttatt ttcattgcaa aaaaaaaaaa aaaaaaaaaa 3480
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540
aaaaaaaaaa aaaaaaaaaa aaaaaactcg gaaggacatg gtgtggaaag tccccaggct 3600
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 3660
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3720
ccaaattaaa gtaacccata acttcgtata gcatacatta tacgaagtta tgaagttcct 3780
attctctaga aagtatagga acttctagtc acctatttca gcatactacg cgcgtagtat 3840
gctgaaatag gtttatcagc acacaatagt ccattatacg cgcgtataat ggcaattgtg 3900
tgctgattgg gttactttaa tttggatccg tcgaccgatg cccttgagag ccttcaaccc 3960
agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt 4020
ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg 4080
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 4140
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 4200
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 4260
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 4320
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 4380
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 4440
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 4500
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4560
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 4620
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 4680
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 4740
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4800
agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4860
acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4920
tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4980
agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 5040
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 5100
agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 5160
cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 5220
ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 5280
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 5340
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 5400
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 5460
tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 5520
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 5580
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 5640
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5700
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5760
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5820
aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5880
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5940
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct 6000
gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg 6060
ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg 6120
gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 6180
ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct 6240
gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt 6300
tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt 6360
tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt 6420
ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg 6480
aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa 6540
gtaatattaa ggtacgtgga ggttttactt gctttaaaaa acctcccaca cctccccctg 6600
aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat 6660
ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 6720
tctagttgtg gtttgtccaa actcatcaat gtatcttatg gtactgtaac tgagctaaca 6780
taa 6783
<210> 45
<211> 6832
<212> DNA
<213> artificial sequence
<220>
<223> 4-6-pGL 2-SS-CMV enhancer-EF 1-UTR1-SecNLuc-2A-eGFP-WPRE-3' UTR (108)
to 120 polyA)-SS*
<400> 45
cccgggaggt accgagctct tacgcgtgct agaattaaag taacccaatc agcacacaat 60
tgccattata cgcgcgtata atggactatt gtgtgctgat aaacctattt cagcatacta 120
cgcgcgtagt atgctgaaat aggtgactag aagttcctat actttctaga gaataggaac 180
ttcataactt cgtataatgt atgctatacg aagttatggg ttactttaat ttggttgctg 240
actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 300
acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg 360
gggactttcc acacccctgg gtcgacgaca ttgattattg actagttatt aatagtaatc 420
aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 480
aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 540
tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 600
gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 660
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 720
tcctacttgg cagtacatct acgtattagt catcgctatt accatggggc agagcgcaca 780
tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga 840
aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag 900
ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg 960
tttgccgcca gaacacagct gccttctccc tcctgtgagt ttggtaagtc gacgggccgg 1020
gcctgggccg ggtccgggcc gggtcgttgg atccccacta cagcccgata ctcaagcttg 1080
acgaattcga gtatccaagg tagtggacta gtgtgacgct gctgacccct ttctttccct 1140
tctgcaggtt ggtgtacagt agcttccaaa ttgattaatt cgagcgaacg cgtcgccacc 1200
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg cctgctcctg 1260
gtgttgcctg ctgccttccc tgccccagtc ttcacactcg aagatttcgt tggggactgg 1320
cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg 1380
tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat 1440
gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg 1500
ggccagatcg aaaaaatttt taaggtggtg taccctgtgg atgatcatca ctttaaggtg 1560
atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc 1620
ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc 1680
ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg 1740
ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcggct 1800
agcgctacta acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct 1860
ggaagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga 1920
cctggatccg gaatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 1980
gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 2040
gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 2100
ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 2160
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 2220
cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 2280
ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 2340
atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 2400
aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 2460
gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 2520
cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 2580
gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 2640
ctgtacaagt aaaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 2700
aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 2760
attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 2820
tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 2880
gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 2940
ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 3000
ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 3060
ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 3120
ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 3180
cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 3240
cgctcgcttt cttgctgtcc aatttctatt aaaggttcct ttgttcccta agtccaacta 3300
ctaaactggg ggatattatg aagggccttg agcatctgga ttctgcctaa taaaaaacat 3360
ttattttcat tgcaagctcg ctttcttgct gtccaatttc tattaaaggt tcctttgttc 3420
cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc 3480
ctaataaaaa acatttattt tcattgcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3540
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3600
aaaaaaaaaa aaaaactcgg aaggacatgg tgtggaaagt ccccaggctc cccagcaggc 3660
agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc 3720
tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caaattaaag 3780
taacccataa cttcgtatag catacattat acgaagttat gaagttccta ttctctagaa 3840
agtataggaa cttctagtca cctatttcag catactacgc gcgtagtatg ctgaaatagg 3900
tttatcagca cacaatagtc cattatacgc gcgtataatg gcaattgtgt gctgattggg 3960
ttactttaat ttggatccgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct 4020
tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc 4080
aactcgtagg acaggtgccg gcagcgctct tccgcttcct cgctcactga ctcgctgcgc 4140
tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4200
acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4260
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4320
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 4380
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 4440
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 4500
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 4560
cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 4620
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 4680
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 4740
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 4800
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 4860
agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 4920
aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 4980
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5040
tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5100
tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5160
tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5220
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5280
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 5340
ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 5400
gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 5460
aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 5520
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 5580
tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 5640
ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 5700
aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 5760
ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 5820
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 5880
agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 5940
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6000
ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg tagcggcgca 6060
ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 6120
gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 6180
caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 6240
cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 6300
tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 6360
acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 6420
gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 6480
ttaacgctta caatttgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 6540
gtgcgggcct cttcgctatt acgccagccc aagctaccat gataagtaag taatattaag 6600
gtacgtggag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 6660
taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 6720
aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 6780
tttgtccaaa ctcatcaatg tatcttatgg tactgtaact gagctaacat aa 6832
<210> 46
<211> 333
<212> DNA
<213> artificial sequence
<220>
<223> three copies of enhancer-1
<400> 46
gggactttcc ggggcggggc acgtggtgca cgggactttc cgtgcacgtg cacgggactt 60
tccgggactt tccgggactt tccgtgcacc acgtggggac tttccgtgca cgggactttc 120
cggggcgggg cacgtggtgc acgggacttt ccgtgcacgt gcacgggact ttccgggact 180
ttccgggact ttccgtgcac cacgtgggga ctttccgtgc acgggacttt ccggggcggg 240
gcacgtggtg cacgggactt tccgtgcacg tgcacgggac tttccgggac tttccgggac 300
tttccgtgca ccacgtgggg actttccgtg cac 333
<210> 47
<211> 1016
<212> DNA
<213> artificial sequence
<220>
<223> chimeric intron
<400> 47
ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 60
cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 120
ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc 180
cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg gtgcgtgcgt 240
gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg 300
cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg 360
cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg 420
cgtggggggg tgagcagggg gtgtgggcgc ggcggtcggg ctgtaacccc cccctgcacc 480
cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 540
gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 600
ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg 660
gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 720
agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 780
cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 840
gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 900
ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 960
ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacag 1016

Claims (135)

1. An expression vector comprising:
(a) A backbone sequence;
(b) A sequence comprising the following components:
(i) An expression cassette comprising a nucleic acid sequence of interest,
(ii) A first target sequence of a first recombinase flanking the 5' side of the expression cassette,
(iii) A second target sequence of the first recombinase flanking the 3' side of the expression cassette, and
(iv) One or more additional target sequences of one or more additional recombinases integrated in the first and the second target sequences in the non-binding region of the first recombinase: and
(c) One or more of the following:
(i) An endonuclease target sequence integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the endonuclease target sequence is located between the backbone sequence and the cleavage sites of the first recombinase and the one or more other recombinases,
(ii) A synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of another enhancer or promoter,
(iii) A Cytomegalovirus (CMV) enhancer integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter,
(iv) Integration of the 5 'untranslated region (5' UTR) comprising the intron between the promoter and the nucleic acid sequence of interest in the expression cassette,
(v) Integrating a vertebrate chromatin insulator in the expression cassette between the nucleic acid of interest and the polyadenylation signal,
(vi) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal,
(vii) Integration of a scaffold/matrix attachment region (S/MAR) located between the nucleic acid of interest and the polyadenylation signal in the expression cassette, or
(viii) A DNA core targeting sequence (DTS) integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the DTS is located between the expression cassette and the cleavage sites of the first recombinase and the one or more other recombinases.
2. The expression vector of claim 1, comprising an endonuclease target sequence integrated within the first and/or second target sequences of the first recombinase and located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the endonuclease target sequence is located between the backbone sequence and the cleavage site of the first recombinase and the one or more other recombinases.
3. The expression vector of claim 1 or 2, comprising an endonuclease target sequence integrated within the first and second target sequences of the first recombinase.
4. The expression vector of any one of claims 1-3, wherein the endonuclease target sequence is for homing an endonuclease.
5. The expression vector of any one of claims 1-4, wherein the endonuclease target sequence is for I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-Dmo I, H-DreI, I-Hmu I, I-Hmu II, I-LlaI, I-MsoI, PI-PkuI, I-PoI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceII, I-SceIII, I-SceIV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-Tli, I-Tlip 061I or I-Vdi 141.
6. The expression vector of any one of claims 1-5, wherein the endonuclease target sequence is for I-SceI.
7. The expression vector of any one of claims 1-5, wherein the endonuclease target sequence is for PI-SceI.
8. The expression vector of any one of claims 1-3, wherein the endonuclease target sequence is for a Cas endonuclease.
9. The expression vector of claim 8, wherein the Cas endonuclease is Cas9.
10. The expression vector of any one of claims 1-9 comprising a synthetic enhancer, wherein the synthetic enhancer comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 12 and is integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of another enhancer or promoter.
11. The expression vector of claim 10, wherein the synthetic enhancer comprises multiple contiguous copies of the sequence set forth in SEQ ID NO:12, at least about 90% identical.
12. The expression vector of claim 11, wherein the synthetic enhancer comprises a sequence that hybridizes to SEQ ID NO:46, and a nucleic acid sequence having at least about 90% identity.
13. The expression vector of any one of claims 10-12, wherein the synthetic enhancer is integrated 5' to the chicken β -actin promoter.
14. The expression vector of claim 13 comprising a chimeric intron comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 47 integrated at the 3 'end of the chicken β -actin promoter and 5' of the nucleic acid sequence of interest.
15. The expression vector of any one of claims 1-14, comprising a CMV enhancer integrated in the expression cassette between the 3 'end of the first target sequence of the first recombinase and the 5' end of the promoter.
16. The expression vector of claim 15, wherein the CMV enhancer is integrated at the 3' end of a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 12 or SEQ ID No. 46.
17. The expression vector of claim 15 or 16, wherein the CMV promoter is integrated 3 'of the CMV enhancer and 5' of the nucleic acid sequence of interest.
18. The expression vector of any one of claims 1-9 comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 or SEQ ID No. 39, said nucleic acid sequence being integrated between the first target sequence of the first recombinase and the nucleic acid sequence of interest.
19. The expression vector of any one of claims 1-9, comprising a 5'utr comprising an intron, wherein the 5' utr is integrated in the expression cassette between the promoter and the nucleic acid sequence of interest.
20. The expression vector of claim 19, wherein the intron comprises a sequence identical to SEQ ID NO:1, and a nucleic acid sequence that is at least about 90% identical.
21. The expression vector of claim 19 or 20, further comprising a non-coding sequence integrated in the intron.
22. The expression vector of claim 20, further comprising a non-coding sequence integrated between two nucleotides in the intron, wherein the two nucleotides correspond to any two nucleotides of positions 25 to 55 of SEQ ID No. 1.
23. The expression vector of claim 21 or 22, wherein the non-coding sequence is S/MAR.
24. The expression vector of claim 23, wherein the S/MAR is MAR-5.
25. The expression vector of claim 19, wherein the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:3, at least about 90% identical.
26. The expression vector of claim 19, wherein the 5' utr comprises a nucleotide sequence that hybridizes to SEQ ID NO:5, and a nucleic acid sequence at least about 90% identical.
27. The expression vector of any one of claims 19-26, wherein the promoter is a chicken β -actin promoter.
28. The expression vector of any one of claims 19-26, wherein the promoter is a CMV promoter.
29. The expression vector of any one of claims 19-28, wherein the promoter is integrated 3' of the CMV enhancer.
30. The expression vector of claim 29, wherein the CMV enhancer is integrated at the 3' end of a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 12 or SEQ ID No. 46.
31. The expression vector of any one of claims 1-30, wherein the polyadenylation signal is integrated 3' of the nucleic acid sequence of interest.
32. The expression vector of claim 31, wherein the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO. 13, SEQ ID NO. 14 or SEQ ID NO. 15.
33. The expression vector of any one of claims 1-30, comprising a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and the polyadenylation signal.
34. The expression vector of claim 33, wherein the vertebrate chromatin insulator is a 5' -HS4 chicken β -globin insulator (cHS 4).
35. The expression vector of any one of claims 1-30 or 33-34, comprising a WPRE located between the nucleic acid of interest and the polyadenylation signal integrated in the expression cassette.
36. The expression vector of any one of claims 1-30 or 33-35, comprising an S/MAR located between the nucleic acid of interest and the polyadenylation signal integrated in the expression cassette.
37. The expression vector of claim 36, wherein the S/MAR is MAR-5.
38. The expression vector of any one of claims 33-37, wherein the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID No. 13, SEQ ID No. 14, or SEQ ID No. 15.
39. The expression vector of any one of claims 2-38, further comprising an enhancer sequence flanking each of the first and second target sequences of the first recombinase.
40. The expression vector of claim 39 comprising at least two enhancer sequences flanking each side of the first and second target sequences of the first recombinase.
41. The expression vector of claim 39 or 40, wherein the enhancer sequence is an SV40 enhancer sequence.
42. The expression vector of any one of claims 1-38, comprising a DTS integrated within the first and/or second target sequences of a first recombinase, located in the non-binding region of the first recombinase and the one or more other recombinases, wherein the DTS is located between the expression cassette and the cleavage site of the first recombinase and the one or more other recombinases.
43. The expression vector of claim 42 wherein said DTS is an SV40 enhancer sequence.
44. The expression vector of claim 42 wherein said DTS is cell specific.
45. The expression vector of any one of claims 1-44, wherein the first and second target sequences and the one or more additional target sequences are selected from the group consisting of a PY54 pal site, an N15 telRL site, a loxP site, a.phi.K02 telRL site, an FRT site, a phiC31 attP site, and a lambda attP site.
46. The expression vector of claim 45, wherein the expression vector contains all of these target sequences.
47. The expression vector of claim 45 or 46, wherein the expression vector comprises a pal site and telRL, loxP and FRT recombinase target binding sequences integrated within the pal site.
48. The expression vector of any one of claims 1-47, wherein the first and second target sequences of the first recombinase each comprise the nucleic acid sequence of SEQ ID NO. 33.
49. The expression vector of any one of claims 1-48, wherein said expression vector is used to produce a bacterial sequence-free vector.
50. The expression vector of claim 49 wherein said bacterial sequence-free vector is a circular covalent closure vector.
51. The expression vector of claim 49 wherein said bacterial sequence-free vector is a linear covalent closure vector.
52. A vector production system comprising a recombinant cell encoding a recombinase under the control of an inducible promoter, wherein the recombinant cell comprises the expression vector of any one of claims 1-51, and wherein the recombinase targets one of the first and second target sequences of the first recombinase or one or more other target sequences of one or more other recombinases in the expression vector.
53. The vector production system of claim 52, wherein said recombinase is TelN, tel, cre or Flp.
54. The vector production system of claim 52 or 53 wherein the recombinant cell further encodes an endonuclease under the control of an inducible promoter, wherein the endonuclease targets the endonuclease target sequence in an expression vector comprising the endonuclease target sequence.
55. The vector production system of claim 54, wherein the endonuclease is a homing endonuclease.
56. The vector production system of claim 55, wherein the homing endonuclease is I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-Dmo I, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfeuI, PI-PkoII, I-PoI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceIII, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-Tli, I-Tlip 061I or I-I141I.
57. The vector production system of any one of claims 54-56, wherein said endonuclease is I-SceI.
58. The vector production system of any one of claims 54-56, wherein said endonuclease is PI-SceI.
59. The vector production system of claim 54 wherein said recombinant cell encodes a nuclease genome editing system comprising an endonuclease.
60. The vector production system of claim 59, wherein the nuclease genome editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nuclease system comprising a guide RNA and a Cas endonuclease.
61. The vector production system of claim 60, wherein the Cas endonuclease is Cas9.
62. The vector production system of any one of claims 52-61, wherein said inducible promoter is heat regulated, chemically regulated, IPTG regulated, glucose regulated, arabinose-inducible, T7 polymerase-regulated, cold shock-inducible, pH-inducible, or a combination thereof.
63. A method of producing a bacterial sequence-free vector comprising incubating the vector production system of any one of claims 52-62 under conditions suitable for expression of a recombinase.
64. The method of claim 63, further comprising incubating the vector production system of any of claims 38-46 under conditions suitable for expression of the endonuclease.
65. The method of claim 63 or 64, further comprising incubating the vector production system of any one of claims 59-62 under conditions suitable for expression of the nuclease genome editing system.
66. The method of any one of claims 63-65, further comprising harvesting the bacterial sequence-free vector.
67. A bacterial-free sequence vector produced by the method of any one of claims 63-66.
68. A bacterial sequence-free vector comprising:
(a) Expression cassette comprising a nucleic acid sequence of interest, and
(b) One or more of the following:
(i) A synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12 located 5' to another enhancer or promoter in the expression cassette,
(ii) A CMV enhancer located 5' to the promoter in the expression cassette,
(iii) Integration of a 5' UTR comprising an intron located between the promoter and the nucleic acid sequence of interest in the expression cassette,
(iv) Integrating a vertebrate chromatin insulator in the expression cassette between the nucleic acid of interest and the polyadenylation signal,
(v) The WPRE located between the nucleic acid of interest and the polyadenylation signal in the expression cassette is integrated,
(vi) Integration of S/MARs in the expression cassette between the nucleic acid of interest and the polyadenylation signal, or
(vii) DTS located 5' to the expression cassette.
69. The bacterial sequence-free vector of claim 68 comprising a synthetic enhancer 5' to another enhancer or promoter in the expression cassette, said synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 12.
70. The bacterial sequence-free vector of claim 69, wherein the synthetic enhancer comprises multiple contiguous copies of a nucleic acid sequence at least about 90% identical to SEQ ID NO. 12.
71. The bacterial sequence-free vector of claim 70, wherein the synthetic enhancer comprises a sequence that hybridizes to SEQ ID NO:46, and a nucleic acid sequence having at least about 90% identity.
72. The bacterial sequence-free vector of any one of claims 69-71, wherein the synthetic enhancer is integrated 5' to the chicken β -actin promoter.
73. The bacterial sequence-free vector of claim 72 comprising a chimeric intron comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO. 47 integrated at the 3 'end of the chicken beta-actin promoter and 5' of the nucleic acid sequence of interest.
74. The bacterial sequence-free vector of claim 68 comprising a CMV enhancer 5' to the promoter in the expression cassette.
75. The bacterial sequence-free vector of claim 74, wherein the CMV enhancer is integrated 3' to a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 12 or SEQ ID No. 46.
76. The bacterial-free sequence vector of claim 74 or 75, wherein the CMV promoter is integrated 3 'of the CMV enhancer and 5' of the nucleic acid sequence of interest.
77. The bacterial sequence-free vector of claim 68 comprising a nucleotide sequence 5' to the nucleic acid sequence of interest that hybridizes to SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38 or SEQ ID NO:39, and a nucleic acid sequence having at least about 90% identity.
78. The bacterial sequence-free vector of claim 68 comprising a 5' UTR comprising an intron between the promoter and the nucleic acid sequence of interest integrated into the expression cassette.
79. The bacterial sequence-free vector of claim 78, wherein the intron comprises a sequence identical to SEQ ID NO:1, and a nucleic acid sequence that is at least about 90% identical.
80. The bacterial sequence-free vector of claim 78 or 79, further comprising a non-coding sequence integrated in the intron.
81. The bacterial-free sequence vector of claim 79, further comprising a non-coding sequence integrated between two nucleotides in the intron, wherein the two nucleotides correspond to any two of the nucleotides at nucleotide positions 25 and 55 of SEQ ID NO. 1.
82. The bacterial sequence-free vector of claim 80 or 81, wherein the non-coding sequence is S/MAR.
83. The bacterial sequence-free vector of claim 82, wherein the S/MAR is MAR-5.
84. The bacterial sequence-free vector of claim 78, wherein the 5' utr comprises a sequence corresponding to SEQ ID NO:3, at least about 90% identical.
85. The bacterial sequence-free vector of claim 78, wherein the 5' utr comprises a sequence corresponding to SEQ ID NO:5, and a nucleic acid sequence at least about 90% identical.
86. The bacterial sequence-free vector of any one of claims 78-85, wherein the promoter is a chicken β -actin promoter.
87. The bacterial sequence-free vector of any one of claims 78-85, wherein the promoter is a CMV promoter.
88. The bacterial sequence-free vector of any one of claims 78-87, wherein the promoter is integrated 3' to the CMV enhancer.
89. The bacterial-free sequence vector of claim 88, wherein the CMV enhancer is integrated 3' to a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID No. 12 or SEQ ID No. 46.
90. The bacterial sequence-free vector of any one of claims 68-89, wherein the polyadenylation signal is integrated 3' to the nucleic acid sequence of interest.
91. The bacterial sequence-free vector of claim 90, wherein the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID No. 13, SEQ ID No. 14 or SEQ ID No. 15.
92. The bacterial-free sequence vector of any one of claims 68-89, comprising a vertebrate chromatin insulator integrated in an expression cassette between a nucleic acid of interest and a polyadenylation signal.
93. The abacterial sequence vector of claim 92, wherein the vertebrate chromatin insulator is cHS4.
94. The bacterial sequence-free vector of any one of claims 68-89 or 92-93, comprising a WPRE between the nucleic acid of interest and the polyadenylation signal integrated in the expression cassette.
95. The bacterial sequence-free vector of any one of claims 68-89 or 92-94, comprising an S/MAR located between the nucleic acid of interest and the polyadenylation signal, integrated in the expression cassette.
96. The bacterial sequence-free vector of claim 95, wherein the S/MAR is MAR-5.
97. The bacterial sequence-free vector of any one of claims 92-96, wherein the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID No. 13, SEQ ID No. 14 or SEQ ID No. 15.
98. The bacterial sequence-free vector of any one of claims 68-97, further comprising flanking enhancer sequences on each side of the expression cassette.
99. The bacterial sequence-free vector of claim 98, comprising at least two enhancer sequences flanking each side of the expression cassette.
100. The bacterial sequence-free vector of claim 98 or 99, wherein the enhancer sequence is an SV40 enhancer sequence.
101. The bacterial sequence-free vector of any one of claims 68-97, comprising DTS located 5' to the expression cassette.
102. The bacterial sequence-free vector of claim 101, wherein the DTS is an SV40 enhancer sequence.
103. The bacterial sequence-free vector of claim 101, wherein the DTS is cell-specific.
104. The bacterial sequence-free vector of any one of claims 68-103 which is a cyclic covalent closure vector.
105. The bacterial sequence-free vector of any one of claims 68-103 which is a linear covalent closed vector.
106. A recombinant cell comprising the expression vector of any one of claims 1-51 or the bacterial sequence-free vector of any one of claims 67-105.
107. A composition comprising the expression vector of any one of claims 1-51 or the bacterial sequence-free vector of any one of claims 67-105.
108. The composition of claim 107, further comprising a delivery agent.
109. The composition of claim 108, wherein the delivery agent is a nanoparticle.
110. The composition of claim 108 or 109, wherein the delivery agent comprises a targeting ligand.
111. The composition of any one of claims 107-110, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
112. A method of treating a disease or disorder in an individual in need thereof, comprising administering to the individual the expression vector of any one of claims 1-51, the bacterial-free sequence vector of any one of claims 67-105, or the pharmaceutical composition of claim 111.
113. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 1.
114. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 2.
115. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 3.
116. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 5.
117. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 12.
118. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 46.
119. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 13.
120. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 14.
121. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 15.
122. The polynucleotide of any one of claims 86-88, further comprising 100-120 adenine nucleotides located 3' of said nucleic acid sequence.
123. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 16.
124. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 17.
125. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 18.
126. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 35.
127. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 36.
128. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 37.
129. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 38.
130. A polynucleotide comprising a nucleic acid sequence that is at least about 90% identical to SEQ ID No. 39.
131. An expression vector comprising the polynucleotide of any one of claims 113-130.
132. An expression vector comprising the polynucleotide of any one of claims 114-116 and the polynucleotide of any one of claims 119-125.
133. A method of gene editing comprising inserting a nucleic acid sequence of interest from the expression vector of any one of claims 1-51, the bacterial-free sequence vector of any one of claims 67-105, or the pharmaceutical composition of any one of claims 107-111 into a target site for gene editing.
134. The method of claim 133, wherein the gene editing is performed by non-homologous end joining.
135. The method of claim 133, wherein the gene editing is performed by homology-directed repair.
CN202280055798.2A 2021-06-16 2022-06-16 Expression vectors, bacterial sequence-free vectors, and methods of making and using the same Pending CN117881788A (en)

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