KR20200071984A - Lentiviral vector system for modulating gene expression using a blue light - Google Patents

Abstract

The present invention relates to a kit for transformation of human somatic cells, capable of regulating expression of a plurality of target genes and using CRISPR interference. The kit for transformation of human somatic cells of the present invention can induce overexpression of several target genes simultaneously in human somatic cells. In particular, since four types of gRNAs can be simultaneously expressed in one vector, it is possible to increase expression rate of the target genes by simplifying a three vector system into a two vector system. Also, regulation of the target gene expression can be made stably.

Description

Lentiviral vector system for modulating gene expression using a blue light}

The present invention relates to a vector system for transforming human somatic cells that can use CRISPR interference and regulate expression of a plurality of target genes.

Cas9 technology, a gene editing technology, has been widely used in the fields of basic science and medical industry, such as gene therapy and production of new transgenic organisms, and new systems are being produced due to technological advances. Recently, a new system for controlling gene expression using blue light has been developed by combining Cas9 mutant (dCas9) without DNA editing function for gene therapy purposes and optogenetic technology (Lauren R Polstein, Charles A Gersbach (2015) A light-inducible CRISPR-Cas9 system for control of endogenous gene activation.Nature chemical biology 11).

However, in the method, the DNA vector containing the dCas9-Optogenesis system is directly introduced into the cell, and according to the induction of expression of the target gene, it is difficult to control the DNA copy every time the vector is introduced into the cell, so the expression level of the target gene is not stable There are disadvantages.

An object of the present invention for solving the problems of the prior art is to provide a vector system capable of stably high-expressing a target gene in human somatic cells.

The problems to be solved by the present invention are not limited to the problems described above, and other problems not described will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention is a first vector comprising a sequence of a gene encoding a biologically inactivated Cas (dCas); And a second vector comprising a sequence of a gene encoding a guide RNA (gRNA) of a target gene to suppress expression in human somatic cells.

As used herein, Cas refers to a CRISPR (Clustered regularly-interspaced short palindromic repeats) related protein.

In a preferred embodiment of the present invention, the dCas may include dCas9.

The first vector may include an Ef1α promoter.

The sequence of the gene encoding the first vector may include the sequence of SEQ ID NO: 21.

In a preferred embodiment of the present invention, the second vector includes an insertion site of a sequence encoding a guide RNA (gRNA) for a target gene; And the Ef1α promoter.

The second vector may include a sequence of a gene encoding gRNA for one or more target genes. For example, the second vector may include a plurality of sgRNAs.

In the above aspect, using the Sangki kit, it is possible to induce overexpression of two or more target genes at the same time. That is, the second vector may include sequences encoding gRNAs for two or more target genes, and they may all operate to simultaneously induce overexpression of two or more target genes.

In a preferred embodiment of the present invention, the target gene may be at least one of INS (Insulin), PCSK1 (proprotein convertase subtilisin/kexin type 1), PCSK2 (proprotein convertase subtilisin/kexin type 2) and GCK (glucokinase) gene .

The target gene is a concept that includes not only the INS, PCSK1, PCSK2, and GCK genes, but also all genes that want to control expression in cells into which the vector is inserted.

In a preferred embodiment of the present invention, the second vector is one of SEQ ID NOs: 1 to 5, one of SEQ ID NOs: 6 to 10, one of SEQ ID NOs: 11 to 15, and one of SEQ ID NOs: 16 to 20 It may contain one sequence. SEQ ID NOs: 1 to 5 include the gRNA sequence targeting the INS gene, SEQ ID NOs: 6 to 10 include the gRNA sequence targeting the PCSK1 gene, and SEQ ID NOs: 11 to 15 target the PCSK2 gene. The gRNA sequence is included, and SEQ ID NOs: 16 to 20 include the gRNA sequence targeting the GCK gene.

In one preferred embodiment of the present invention, the second vector may include one of SEQ ID NOs: 22 to 26.

The kit for transformation of human somatic cells of the present invention can induce overexpression of several target genes simultaneously in human somatic cells.

In particular, in the kit for transformation of human somatic cells of the present invention, since 4 types of gRNAs can be simultaneously expressed in one vector, it is possible to increase the expression rate of the target gene by simplifying 3 vector systems to 2 vector systems, Gene expression regulation can be stably achieved.

1 is a view showing a process of cloning the four kinds of gRNA produced in the present invention into a plasmid.
Figure 2 is a diagram showing the construction and cloning process of dCas9-photogenetic DNA vector.
3 is a view showing the manufacturing process and structure of the first vector of the present invention.
4A and 4B are diagrams illustrating a manufacturing process and structure of a second vector of the present invention.
5 is a diagram showing the manufacturing process of human foreskin Fibrolast Cells (hFFs) and human skin fibroblasts (HDFs) overexpressing hINS, hGCK, hPCSK1 and hPCK2 genes.
6 is a diagram confirming whether the target gene is expressed when the first vector and the second vector are expressed.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Preparation of ingredients

Plasmids are phH1-gRNA (#53186), pmU6-gRNA (#53187), phU6-gRNA (#53188), ph7SK-gRNA (#53189), pLV hUbC-VP64 dCas9 VP64-T2A-GFP (#59791) pcDNA3. 1-CMV ^P -CibN-dCas9-CibN (#60553), pcDNA3.1-Ef1α ^P -Cyr2FL-VP64 (#60554), Lenti-dCas9-KRAB-blast(#89567), lenti-EF1a-dCas9-KRAB- Puro #99372), pMD2.G (#12259), and psPAX2 (#12260) plasmids were purchased from Addgene. .

Guide RNA sequence design and fabrication were performed using http://crispr-era.stanford.edu/ .

A primer for gene expression verification was prepared and used, and the sequences are shown in tables in each example.

Enzymes are SpeⅠ(NEB), SacⅠ(NEB), NruⅠ(NEB), MluⅠ(NEB), XbaⅠ(NEB), BsrGⅠ(NEB), BbsI(Thermo scientific), BsmBI (Thermo scientific), T4 DNA ligase(NEB) Was used.

For virus production, 293T cells, Superfect transfection reagent (Qiagen, 301305), Amicon Ultra-15 Centrifugal Filter (Merck millipore, UFC910024), and Polybrene (Merck millipore, TR-1003-G) were used.

Human Foreskin Fibroblast (hFF) and human Dermal Fibroblast (hDF) were used as cells for overexpression.

Production and cloning of gRNA

After designing 5 pairs of oligonucleotides for hINS, hGCK, hPCSK1 and hPCSK2 genes, each was designed to contain a BbsI restriction enzyme recognition site. The produced 20 gRNA sequences are shown in [Table 1].

[Table 1]

Each of the forward and reverse sequences of the gRNA oligonucleotides listed in [Table 1] was put in an experimental 1.5 ml tube in the same amount (1:1) and reacted at 95° C. for 5 minutes, gradually lowering the temperature until it reached room temperature. Double strands were formed between enemy DNA.

There are nucleotide sequences that can be cut using the restriction enzyme BbsI at the gRNA positions of the phH1-gRNA, pmU6-gRNA, phU6-gRNA and ph7SK-gRNA plasmids. Thus, a truncated pure plasmid was obtained using a Gel elution kit, and each double-stranded gRNA obtained above was cut using the restriction enzyme BbsI to clone the gRNA into the plasmid. The truncated four gRNAs were ligated into the plasmid backbone containing the promoter and the rest of the long chimeric gRNA using T4 ligase enzyme (FIG. 1).

Decas 9-Photogenetic DNA vector production and cloning

3-1. pcDNA3.1-int_Ef1α ^P -CibN -dCas9-CibN production

To amplify the Ef1α ^P (hereinafter int_Ef1α ^P ) containing the intronA region of the #99372 vector, a primer was attached with SpeⅠ/ SacⅠ linker (Table 2), and then subjected to Herculase II PCR (agilent), followed by DNA gel with partial digestion By elution (FAVORGEN), only the desired site was obtained.

[Table 2]

Int_Ef1α ^P was introduced into pJET1.2/blunt (Thermo) using XL10-Gold®Ultracompetent Cells (STRATAGENE), mini prep (GeneAll), and then int_Ef1α cut and amplified by restriction enzymes SpeI (NEB) and SacI (NEB) ^P was obtained.

CMV ^P of pcDNA3.1-CibN-dCAS9-CibN (addgene #60553) vector was partially digested with restriction enzymes SpeⅠ and SacⅠ and removed using CM4 ^P removed #60553 vector using T4 ligase (Thermo). by connecting the ^P int_Ef1α Cas 9-light genetics DNA vector ^{ⅰ (pcDNA3.1-int_Ef1α P -CibN -dCas9} -CibN) was produced to (Figure 2a).

3-2. pcDNA3.1-BsmBⅠ-int_Ef1α ^P -Cyr2FL-VP64 production

pLV hUbC-VP64 dCas9 VP64-T2A-GFP (addgene #59791) To amplify the BsmBI site (hereinafter BsmBⅠ) of the vector, a primer was attached with NruI/MluI linker (Table 3) and Herculase II PCR was performed, followed by DNA gel By elution, only the desired site was obtained.

[Table 3]

BsmBI was introduced into pJET1.2/blunt using XL10-Gold®Ultracompetent Cells, and mini-prep to obtain BsmBI amplified by restriction enzymes NruI (NEB) and MluI (NEB). The pcDNA3.1-Cry2FL-VP64 (addgene #60554) vector was cut with restriction enzymes NruI and MluI, and then Tsm ligase was used to connect BsmBI to produce decas 9-optogenetic DNA vector II (FIG. 2B).

Production and cloning of Decas 9-optogenetic lentiviral vector

4-1. pcDNA3.1-int_Ef1α ^P -CibN -dCas9-CibN(addgene #60553) + lenti-EF1a-dCas9-KRAB-Puro(addgene #99372)

In order to amplify the Ef1α ^P- CibN-dCas9-CibN region of #60553 vector cloned in Example 3-1, a primer was attached with XbaⅠ/BsrGⅠ linker (Table 4) and Herculase II PCR was performed, followed by DNA gel elution. To obtain only the desired site.

[Table 4]

Ef1α ^P- CibN-dCas9-CibN site is introduced into pJET1.2/blunt using XL10-Gold® Ultracompetent Cells. After Miniprep, Ef1α ^P- CibN-dCas9-CibN sites amplified by restriction enzymes XbaI (NEB) and BsrGI (NEB) were obtained.

After cutting the Lenti-Ef1α-dCas9-KRAB-Puro (addgene #99372) vector with restriction enzymes NheⅠ and BsrGⅠ, T4 ligase was used to connect the Ef1α ^P -CibN-dCas9-CibN site to decas 9-photogenetic lent Virus vector I (first vector) was produced (Fig. 3).

4-2. pcDNA3.1-BsmBⅠ-int_Ef1α ^P -Cyr2FL-VP64 (addgene #60554) + Lenti-dCas9-KRAB-blast (addgene #89567)

In order to amplify the BsmBⅠ-Ef1α ^P- CRY2FL-VP64 region of the #60554 vector cloned in Example 3-2, primers were attached with XbaⅠ/BsrGⅠ linkers (Table 5) and then subjected to Herculase II PCR, followed by DNA gel elution. To obtain only the desired site.

[Table 5]

In XL10-Gold®Ultracompetent pJET1.2 / blunt using Cells Ef1α ^P--CibN dCas9 CibN-introducing portion, and a restriction enzyme after Mini prep XbaⅠ (NEB), BsrGⅠ (NEB) a cut-amplified by BsmBⅠ Ef1α ^P -CRY2FL-VP64 site was obtained.

The Lenti-dCas9KRAB-blast (addgene #89567) vector was cut with restriction enzymes NheⅠ and BsrGⅠ, and then Tsm ligase was used to connect the BsmBⅠ-Ef1α ^P -CRY2FL-VP64 region to decas 9-photogenetic lentivirus vector Ⅱ. It was produced (Fig. 4a).

Construction of lentiviral vector introduced with gRNA

Golden Gate assembly was performed by simultaneously inserting T4 DNA ligase and BsmBI restriction enzyme to insert the gRNA plasmid having four different promoters prepared in Example 2 into the final lentiviral vector according to Set1-5. Table 6].

[Table 6]

Through the above process, a lentiviral vector (second vector) in which four gRNAs are expressed by four different promoters in one vector was finally constructed (FIG. 4B ).

Production of lentivirus

The first vector prepared in Example 4 (Lenti-int_Ef1αP-CibN-dCas9-CibN-Puro) and the second vector prepared in Example 5 (Lenti-4gRNA-int_Ef1αP-Cyr2FL-VP64-BSD) Set 1-5, respectively 293T cells were transfected using a transfection reagent (Superfect) together with lentiviral packaging vectors pMD2.G and psPAX2. After 6 hours, the cells were changed to serum-free expression media, and after 26 hours, 38 hours, and 50 hours of transfection, samples obtained by collecting expression media containing viruses were stored at -80°C. Using Amicon Ultra-15 Centrifugal Filter, a large volume of virus was concentrated to about 250 ul to undergo transformation or dispensing and stored at -80°C.

Construction of human FF, DF cells overexpressing blue light-mediated hINS, hGCK, hPCSK1, and hPCK2 genes

Human Foreskin Fibrolast Cells (hFFs), Human Derma Fibrolast Cells (hDFs) int_Ef1αP-CibN-dCas9-CibN lentivirus and 4gRNA-int_Ef1αP-Cyr2FL-VVP64 virus produced in Example 5 Was transduced to make hVE, hGCK, hPCSK1 and hPCK2 gene overexpression mVEC. To increase the infection efficiency, after changing to a medium to which polybrene (10 ug/ml) was added, about 20-50 ul of concentrated virus was added. When CibN-dCas9-CibN was expressed, transduction was confirmed by using puromycin and Cyr2FL-VP64 expression as blasticidin as a selective marker (FIG. 5).

Lenti-4gRNA-int_Ef1αP-Cyr2FL-VP64-BSD(#89567+#60554) and Lenti-int_Ef1αP-CibN -dCas9-CibN-Puro (#99372+#60553) containing gRNA Set 1~5 using restriction enzyme SpeⅠ After cutting it, electrophoresis was performed to confirm the desired size of DNA (FIG. 6).

<110> Industry-Academic Cooperation Foundation, Konkuk University <120> Lentiviral vector system for modulating gene expression using a blue light <130> 1064988 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 1 <400> 1 ttgtttgacc ccaggcccta atgggcc 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 2 <400> 2 ttgtttgcta atgacccgct ggtcctg 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 3 <400> 3 ttgtttgtga gactataaag ccagcgg 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 4 <400> 4 ttgtttgctc tgagactata aagccag 27 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hINS gRNA 5 <400> 5 ttgtttggga agaggtgctg acgacca 27 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 1 <400> 6 caccgagagt atttgagcag ccgcc 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 2 <400> 7 caccggcccg cacaaggctg aggaa 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 3 <400> 8 caccgtagcg gtcactgtag tgaca 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 4 <400> 9 caccgagtgg gggattctcc tgcca 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hGCK gRNA 5 <400> 10 caccggatcc tctgctcatc agaac 25 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 1 <400> 11 tcccaagctg ctgggactcg cgacc 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 2 <400> 12 tcccaatcta tccccgcgac tctct 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 3 <400> 13 tcccacgcga ctctctaggg agctc 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 4 <400> 14 tcccacgcgg agtagcctgg gaagc 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK1 gRNA 5 <400> 15 tcccacttgg tagttatgtt caagt 25 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 1 <400> 16 cctcggccgc tggcctgatt acagc 25 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 2 <400> 17 cctcgtgcgc tgcgccccca gaagg 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 3 <400> 18 cctcgggcgg gcccggggcg gccca 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 4 <400> 19 cctcgccgcc ctcattggcc gctgg 25 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hPCSK2 gRNA 5 <400> 20 cctcggcccg gggcggccca gggcg 25 <210> 21 <211> 15239 <212> DNA <213> Artificial Sequence <220> <223> lentivirus vector 1 <400> 21 aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga 60 gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag 120 tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag 180 gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga 240 cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc 300 tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc 360 tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt 420 ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta 480 ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta 540 acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga 600 atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa 660 caaattggct gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa 720 gaatagtttt tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat 780 cgtttcagac ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag 840 aaggtggaga gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg 900 cgccaattct gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt 960 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa 1020 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga 1080 gatccagttt ggttaattag ctagcagata tacgcgttga cattgattat tgactagtct 1140 aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc 1200 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt 1260 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 1320 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 1380 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt 1440 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg 1500 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc 1560 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc 1620 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac 1680 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca 1740 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat 1800 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag 1860 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg 1920 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg 1980 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca 2040 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc 2100 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt 2160 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta 2220 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt 2280 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt 2340 gacgtgagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac 2400 gactcactat agggagaccc aagctggcta gcgccatgga ctacaaagac catgacggtg 2460 attataaaga tcatgacatc gatataaatg gagctatagg aggtgacctt ttgctcaatt 2520 ttcctgacat gtcggtccta gagcgccaaa gggctcacct caagtacctc aatcccacct 2580 ttgattctcc tctcgccggc ttctttgccg attcttcaat gattaccggc ggcgagatgg 2640 acagctatct ttcgactgcc ggtttgaatc ttccgatgat gtacggtgag acgacggtgg 2700 aaggtgattc aagactctca atttcgccgg aaacgacgct tgggactgga aatttcaaga 2760 aacggaagtt tgatacagag actaaggatt gtaatgagaa gaagaagaag atgacgatga 2820 acagagatga cctagtagaa gaaggagaag aagagaagtc gaaaataaca gagcaaaaca 2880 atgggagcac aaaaagcatc aagaagatga aacacaaagc caagaaagaa gagaacaatt 2940 tctctaatga ttcatctaaa gtgacgaagg aattggagaa aacggattat attcatcacg 3000 attacaagga tgacgatgac aagatggccc ccaagaagaa gaggaaggtg ggccgcggaa 3060 tggataagaa atactcaata ggcttagcta tcggcacaaa tagcgtcgga tgggcggtga 3120 tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc 3180 acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag 3240 cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt 3300 atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac 3360 ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa 3420 atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa 3480 aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata 3540 tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat aatagtgatg 3600 tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta 3660 ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac 3720 gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta tttgggaatc 3780 tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag 3840 atgctaaatt acagctttca aaagatactt acgatgatga tttagataat ttattggcgc 3900 aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt 3960 tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa 4020 tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct ttagttcgac 4080 aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac ggatatgcag 4140 gttatattga tgggggagct agccaagaag aattttataa atttatcaaa ccaattttag 4200 aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat ttgctgcgca 4260 agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt gagctgcatg 4320 ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt gagaagattg 4380 aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt ggcaatagtc 4440 gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat tttgaagaag 4500 ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac tttgataaaa 4560 atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat tttacggttt 4620 ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca gcatttcttt 4680 caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga aaagtaaccg 4740 ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt gttgaaattt 4800 caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg ctaaaaatta 4860 ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag gatattgttt 4920 taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa acatatgctc 4980 acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact ggttggggac 5040 gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa acaatattag 5100 attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc catgatgata 5160 gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc gatagtttac 5220 atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt ttacagactg 5280 taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa aatatcgtta 5340 ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg cgagagcgta 5400 tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa gagcatcctg 5460 ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa aatggaagag 5520 acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat gtcgatgcca 5580 ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta acgcgttctg 5640 ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa aagatgaaaa 5700 actattggag acaacttcta aacgccaagt taatcactca acgtaagttt gataatttaa 5760 cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc aaacgccaat 5820 tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt cgcatgaata 5880 ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc ttaaaatcta 5940 aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag attaacaatt 6000 accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg attaagaaat 6060 atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat gttcgtaaaa 6120 tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc ttttactcta 6180 atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt cgcaaacgcc 6240 ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg cgagattttg 6300 ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa acagaagtac 6360 agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac aagcttattg 6420 ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca acggtagctt 6480 attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta aaatccgtta 6540 aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat ccgattgact 6600 ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa ctacctaaat 6660 atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc ggagaattac 6720 aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat ttagctagtc 6780 attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg tttgtggagc 6840 agcataagca ttatttagat gagattattg agcaaatcag tgaattttct aagcgtgtta 6900 ttttagcaga tgccaattta gataaagttc ttagtgcata taacaaacat agagacaaac 6960 caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat cttggagctc 7020 ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg tctacaaaag 7080 aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa acacgcattg 7140 atttgagtca gctaggaggt gacccaattg ccggatccaa ggctagcccg aaaaagaaac 7200 gcaaagttgg gcgcgccacc atgaatggag ctataggagg tgaccttttg ctcaattttc 7260 ctgacatgtc ggtcctagag cgccaaaggg ctcacctcaa gtacctcaat cccacctttg 7320 attctcctct cgccggcttc tttgccgatt cttcaatgat taccggcggc gagatggaca 7380 gctatctttc gactgccggt ttgaatcttc cgatgatgta cggtgagacg acggtggaag 7440 gtgattcaag actctcaatt tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac 7500 ggaagtttga tacagagact aaggattgta atgagaagaa gaagaagatg acgatgaaca 7560 gagatgacct agtagaagaa ggagaagaag agaagtcgaa aataacagag caaaacaatg 7620 ggagcacaaa aagcatcaag aagatgaaac acaaagccaa gaaagaagag aacaatttct 7680 ctaatgattc atctaaagtg acgaaggaat tggagaaaac ggattatatt tgtacaggtt 7740 caggggaggg aagaggttcc ctgctcacat gcggagacgt agaagagaat cccggaccca 7800 tgacctctag agagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg 7860 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc 7920 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc 7980 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc 8040 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga 8100 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca 8160 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc 8220 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct 8280 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca 8340 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg 8400 cctgagaatt cgatatcaag cttatcggta atcaacctct ggattacaaa atttgtgaaa 8460 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 8520 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 8580 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 8640 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 8700 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 8760 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 8820 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 8880 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 8940 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 9000 tttgggccgc ctccccgcat cgataccgtc gacctcgaga cctagaaaaa catggagcaa 9060 tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg 9120 aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg 9180 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 9240 cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg 9300 attggcagaa ctacacacca gggccaggga tcagatatcc actgaccttt ggatggtgct 9360 acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga gagaacaccc 9420 gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtattagagt 9480 ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat ccggactgta 9540 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc 9600 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt 9660 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta 9720 gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 9780 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 9840 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 9900 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 9960 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 10020 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 10080 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 10140 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 10200 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 10260 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 10320 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 10380 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 10440 tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 10500 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 10560 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 10620 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 10680 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 10740 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 10800 tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca attaatcatc 10860 ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 10920 gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 10980 cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 11040 cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 11100 ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 11160 gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 11220 ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 11280 ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 11340 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 11400 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 11460 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 11520 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc 11580 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 11640 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 11700 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 11760 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 11820 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 11880 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 11940 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 12000 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 12060 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 12120 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 12180 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 12240 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 12300 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 12360 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 12420 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 12480 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 12540 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 12600 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 12660 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 12720 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 12780 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 12840 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 12900 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 12960 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 13020 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 13080 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 13140 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 13200 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 13260 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 13320 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 13380 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 13440 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 13500 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 13560 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 13620 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 13680 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 13740 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat 13800 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 13860 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 13920 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 13980 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 14040 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 14100 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 14160 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 14220 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 14280 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 14340 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 14400 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 14460 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 14520 ggtaggcgtg tacggtggga ggtctatata agcagcgcgt tttgcctgta ctgggtctct 14580 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 14640 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 14700 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc 14760 ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg caggactcgg 14820 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt 14880 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg 14940 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa 15000 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg 15060 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca 15120 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa 15180 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaa 15239 <210> 22 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> lentivirus vector 2 (Set1_4gRNA) <400> 22 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700 aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgacc ccaggcccta atgggccgtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacgg cggctgctca 3240 aatactctcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 tataagttct gtatgagacc actctttccc aagctgctgg gactcgcgac cgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acgctgtaat 3960 caggccagcg gccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560 cgaaaagtgc cacctgac 13578 <210> 23 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> lentivirus vector 2 (Set2_4gRNA) <400> 23 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700 aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgcta atgacccgct ggtcctggtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactt cctcagcctt 3240 gtgcgggccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 tataagttct gtatgagacc actctttccc aatctatccc cgcgactctc tgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acccttctgg 3960 gggcgcagcg cacgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560 cgaaaagtgc cacctgac 13578 <210> 24 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> lentivirus vector 2 (Set3_4gRNA) <400> 24 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700 aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgtga gactataaag ccagcgggtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactg tcactacagt 3240 gaccgctacg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 tataagttct gtatgagacc actctttccc acgcgactct ctagggagct cgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa actgggccgc 3960 cccgggcccg cccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560 cgaaaagtgc cacctgac 13578 <210> 25 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> lentivirus vector 2 (Set4_4gRNA) <400> 25 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700 aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttgctc tgagactata aagccaggtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaactg gcaggagaat 3240 cccccactcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 tataagttct gtatgagacc actctttccc acgcggagta gcctgggaag cgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa acccagcggc 3960 caatgagggc ggcgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560 cgaaaagtgc cacctgac 13578 <210> 26 <211> 13578 <212> DNA <213> Artificial Sequence <220> <223> lentivirus vector 2 (Set5_4gRNA) <400> 26 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 360 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 540 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1140 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1440 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1560 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1620 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2280 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2460 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2580 agatccagtt tggttaatta gctagcttag ggttaggcgt tttgcgctgc ttcgcgatta 2640 cagggacagc agagatccag tttggttaat taaataactt cgtatagcat acattatacg 2700 aagttatgat agatccgacg ccgccatctc taggcccgcg ccggccccct cgcacagact 2760 tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata tttcctagta 2820 actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa tactagctac 2880 attttacatg ataggcttgg atttctataa gagatacaaa tactaaatta ttattttaaa 2940 aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt gagactataa 3000 atatcccttg gagaaaagcc ttgtttggga agaggtgctg acgaccagtt ttagagctag 3060 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 3120 tgcttttttt gtacgctcag cggagaaaaa aagcaccgac tcggtgccac tttttcaagt 3180 tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacgt tctgatgagc 3240 agaggatccg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3300 tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3360 cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3420 caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3480 catgggaaat aggccctctt cctgcccgac cttggatcga tccgcctgaa cgctgacgtc 3540 atcaacccgc tccaaggaat cgcgggccca gtgtcactag gcgggaacac ccagcgcgcg 3600 tgcgccctgg caggaagatg gctgtgaggg acaggggagt ggcgccctgc aatatttgca 3660 tgtcgctatg tgttctggga aatcaccata aacgtgaaat gtctttggat ttgggaatct 3720 tataagttct gtatgagacc actctttccc acttggtagt tatgttcaag tgttttagag 3780 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 3840 tcggtgcttt ttttggtcat cagtccgaga aaaaaagcac cgactcggtg ccactttttc 3900 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa accgccctgg 3960 gccgccccgg gccgaggtac ccaagcggcg cacaagctat ataaacctga aggaagtctc 4020 aactttacac ttaggtcaag ttgcttatcg tactagagct tcagcaggaa atttaactaa 4080 aatctaattt aaccagcata gcaaatatca tttattccca aaatgctaaa gtttgagata 4140 aacggacttg atttccggct gttttgacac tatccagaat gccttgcaga tgggtggggc 4200 atgctaaata ctgcagacaa ttaattaacc cgtgtcggct ccagatctgg cctccgcgcc 4260 gggttttggc gcctcacgcg ttgacattga ttattgacta gtctaggtct tgaaaggagt 4320 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 4380 gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg 4440 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 4500 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt 4560 aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc 4620 ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc gggttggaag 4680 tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg cttgagttga 4740 ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct 4800 cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg cgacgctttt 4860 tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta tttcggtttt 4920 tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg 4980 cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc ggcctgctct 5040 ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc 5100 ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag ggagctcaaa 5160 atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa ggaaaagggc 5220 ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc cgtccaggca 5280 cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg aggggtttta 5340 tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag cttggcactt 5400 gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca ttctcaagcc 5460 tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgacgtg agctctctgg 5520 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 5580 acccaagctg gctagcgcca tggactacaa agaccatgac ggtgattata aagatcatga 5640 catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga aggtgggccg 5700 catgaagatg gacaaaaaga ctatagtttg gtttagaaga gacctaagga ttgaggataa 5760 tcctgcatta gcagcagctg ctcacgaagg atctgttttt cctgtcttca tttggtgtcc 5820 tgaagaagaa ggacagtttt atcctggaag agcttcaaga tggtggatga aacaatcact 5880 tgctcactta tctcaatcct tgaaggctct tggatctgac ctcactttaa tcaaaaccca 5940 caacacgatt tcagcgatct tggattgtat ccgcgttacc ggtgctacaa aagtcgtctt 6000 taaccacctc tatgatcctg tttcgttagt tcgggaccat accgtaaagg agaagctggt 6060 ggaacgtggg atctctgtgc aaagctacaa tggagatcta ttgtatgaac cgtgggagat 6120 atactgcgaa aagggcaaac cttttacgag tttcaattct tactggaaga aatgcttaga 6180 tatgtcgatt gaatccgtta tgcttcctcc tccttggcgg ttgatgccaa taactgcagc 6240 ggctgaagcg atttgggcgt gttcgattga agaactaggg ctggagaatg aggccgagaa 6300 accgagcaat gcgttgttaa ctagagcttg gtctccagga tggagcaatg ctgataagtt 6360 actaaatgag ttcatcgaga agcagttgat agattatgca aagaacagca agaaagttgt 6420 tgggaattct acttcactac tttctccgta tctccatttc ggggaaataa gcgtcagaca 6480 cgttttccag tgtgcccgga tgaaacaaat tatatgggca agagataaga acagtgaagg 6540 agaagaaagt gcagatcttt ttcttagggg aatcggttta agagagtatt ctcggtatat 6600 atgtttcaac ttcccgttta ctcacgagca atcgttgttg agtcatcttc ggtttttccc 6660 ttgggatgct gatgttgata agttcaaggc ctggagacaa ggcaggaccg gttatccgtt 6720 ggtggatgcc ggaatgagag agctttgggc taccggatgg atgcataaca gaataagagt 6780 gattgtttca agctttgctg tgaagtttct tctccttcca tggaaatggg gaatgaagta 6840 tttctgggat acacttttgg atgctgattt ggaatgtgac atccttggct ggcagtatat 6900 ctctgggagt atccccgatg gccacgagct tgatcgcttg gacaatcccg cgttacaagg 6960 cgccaaatat gacccagaag gtgagtacat aaggcaatgg cttcccgagc ttgcgagatt 7020 gccaactgaa tggatccatc atccatggga cgctccttta accgtactca aagcttctgg 7080 tgtggaactc ggaacaaact atgcgaaacc cattgtagac atcgacacag ctcgtgagct 7140 actagctaaa gctatttcaa gaacccgtga agcacagatc atgatcggag cagcacctga 7200 tgagattgta gcagatagct tcgaggcctt aggggctaat accattaaag aacctggtct 7260 ttgcccatct gtgtcttcta atgaccaaca agtaccttcg gctgttcgtt acaacgggtc 7320 aaagagagtg aaacctgagg aagaagaaga gagagacatg aagaaatcta ggggattcga 7380 tgaaagggag ttgttttcga ctgctgaatc ttcttcttct tcgagtgtgt ttttcgtttc 7440 gcagtcttgc tcgttggcat cagaagggaa gaatctggaa ggtattcaag attcatctga 7500 tcagattact acaagtttgg gaaaaaatgg ttgcaaacca attgccggat ccaaggctag 7560 cccgaaaaag aaacgcaaag ttgggcgcgc cgacgcgctg gacgatttcg atctcgacat 7620 gctgggttct gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga 7680 tgactttgat ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt 7740 aattaactac ccgtacgacg ttccggacta cgcttcttgt acaggcagtg gagagggcag 7800 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg ccaagccttt 7860 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat 7920 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg 7980 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc 8040 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg 8100 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa 8160 agccatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc 8220 ctctggttat gtgtgggagg gctaagaatt cgatatcaag cttatcggta atcaacctct 8280 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct 8340 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat 8400 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt 8460 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat 8520 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc 8580 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga 8640 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc 8700 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga 8760 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc 8820 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacctcgaga 8880 cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc 8940 ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt 9000 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 9060 actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca 9120 cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc 9180 actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc 9240 caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc 9300 ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg 9360 agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct 9420 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca 9480 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 9540 gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc 9600 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 9660 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 9720 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 9780 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 9840 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg 9900 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 9960 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 10020 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 10080 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 10140 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 10200 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 10260 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg 10320 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 10380 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 10440 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 10500 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 10560 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 10620 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca 10680 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg 10740 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc 10800 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac 10860 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg 10920 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag 10980 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc 11040 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac 11100 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc 11160 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc 11220 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt 11280 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 11340 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct 11400 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 11460 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 11520 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 11580 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 11640 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 11700 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 11760 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 11820 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 11880 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 11940 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 12000 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 12060 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 12120 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 12180 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 12240 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 12300 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 12360 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 12420 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 12480 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 12540 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 12600 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 12660 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 12720 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 12780 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 12840 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 12900 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 12960 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 13020 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 13080 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 13140 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 13200 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 13260 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 13320 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 13380 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 13440 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 13500 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 13560 cgaaaagtgc cacctgac 13578

Claims (10)

A kit for transformation of human somatic cells,
The human somatic cells are human foreskin fibrolast cells (hFFs) or human skin fibroblasts (Human Derma Fibrolast Cells; hDFs),
The kit,
A first vector comprising a sequence of a gene encoding a biologically inactivated Cas (dCas); And
A kit for transformation of human somatic cells, comprising a second vector comprising a sequence of a gene encoding a guide RNA (gRNA) of a target gene to induce overexpression in human somatic cells.
According to claim 1,
The dCas, dCas9 containing, kit for transformation of human somatic cells.
According to claim 1,
The first vector comprises the Ef1α promoter, a kit for transformation of human somatic cells.
According to claim 1,
The sequence of the gene encoding the first vector, consisting of the sequence of SEQ ID NO: 21, kit for transformation of human somatic cells.
According to claim 1,
The second vector includes an insertion site of a sequence encoding a guide RNA (gRNA) for a target gene; And
Kit for transformation of human somatic cells, comprising the Ef1α promoter.
According to claim 1,
A kit for transformation of human somatic cells, wherein the second vector comprises a sequence of a gene encoding a gRNA for one or more target genes.
According to claim 1,
The kit is to induce overexpression of two or more target genes at the same time, a kit for transformation of human somatic cells.
According to claim 1,
The target gene comprises one or more of INS, PCSK1, PCSK2 and GCK genes, kit for transformation of human somatic cells.
The method of claim 8,
The gRNA targeting the INS gene includes one of SEQ ID NOs: 1 to 5,
The gRNA targeting the PCSK1 gene includes one of SEQ ID NOs: 6 to 10,
The gRNA targeting the PCSK2 gene includes one of SEQ ID NOs: 11 to 15,
The gRNA targeting the GCK gene comprises the sequence of one of SEQ ID NOs: 16 to 20, kit for transformation of human somatic cells.
According to claim 1,
The second vector comprises the sequence of one of SEQ ID NO: 22 to 26, kit for transformation of human somatic cells.

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