KR101557974B1 - Vectors for Preparing Serotype 6 Recombinant Adenovirus - Google Patents

Abstract

The present invention relates to a vector for producing serotype 6 recombinant adenovirus, specifically, a serotype 6 adenovirus genome which does not contain the E1 region, and a serotype 6 recombinant adenovirus preparation comprising a gene encoding a target protein A rescue vector for preparing serotype 6 recombinant adenovirus comprising the serotype 6 adenovirus genome from which the shuttle vector and the El region have been removed, and a serotype 6 adenovirus which homologously recombines the shuttle vector and the rescue vector. The present invention relates to a method for producing a virus.

Description

{Vectors for Preparing Serotype 6 Recombinant Adenovirus}

The present invention relates to a vector for producing serotype 6 recombinant adenovirus, and more specifically, to a serotype 6 recombinant adenovirus comprising a serotype 6 adenovirus genome which does not contain the E1 region and a gene encoding a target protein Shuttle vector for manufacturing; A rescue vector for the preparation of serotype 6 recombinant adenovirus comprising the serotype 6 adenovirus genome from which the El region has been removed; And a method for producing serotype 6 adenovirus, wherein the shuttle vector and the rescue vector are homologous recombined.

The gene transfer technology is largely classified into a viral vector-based transfer method, a non-viral delivery method using a synthetic phospholipid or a synthetic cationic polymer, and a temporary electrical stimulation And electroporation in which a gene is introduced by adding the gene. Among them, the method using the viral transporter is the most preferred method because the transfer of the gene can be efficiently performed. Viruses used as viral vectors or viral vectors include retroviral vectors (lentiviral vectors) and adenoviral vectors (adeno-associated viral vectors), as well as herpes simplex viral vectors ), Alpha viral vector, and the like.

 Retroviruses are easy to proliferate, infect various cells and are harmless to the human body, but are difficult to infect cells after mitosis and are difficult to transfer in vivo. In addition, it integrates into the genome of the host cell, which can inhibit or necrotize the function of normal cells and have a disadvantage of being mutated when integrated into a proto-oncogene. Adenoviruses, on the other hand, have high transduction efficiency and are well infected even during replication or differentiation and are not integrated (inserted) into host cells.

 Methods for producing recombinant adenovirus are as follows. 1) direct cloning into adenovirus genome, 2) homologous recombination in adenovirus producing cell line using shuttle vector and rescue vector, 3) There is a method of homologous recombination in Escherichia coli (for example, BJ5183) having a recombinase activity using a vector and a rescue vector. Currently, method 3) has been used to produce serotype 2 or serotype 5 recombinant adenoviruses. The transduction efficiency (TE) of the target gene mediated by the adenovirus is blocked by a neutralizing antibody (NAb) present in vivo. Therefore, it is important to deliver the target gene to the serotype adenovirus corresponding to the type of neutralizing antibody that exists at least in the serum of the subject or species.

There are about 55 serotypes of known human adenoviruses, and serotype 2 or 5 adenovirus (Ad5) belonging to subgroup C has been most widely used for gene transfer, gene therapy, cell therapy and vaccine development. Serotype 2 adenoviruses are highly likely to cause an immune response. On the other hand, most people (eg sub-Saharan African) have difficulty in obtaining the clinical therapeutic efficacy anticipated by neutralizing antibodies (NAbs) that have already been exposed to Ad5 (Abbink P et al ., J Virol. 2007, 81 (9): 4654-4663). Serologic type 3 (91.1%), serotype 2 (37.5%), serotype 1 (27.1%), serotype 7 (7%) and serotype 2 were found to be serotypes 1 and 2 in children with cystic fibrosis Piedra PA et al ., Pediatrics 1998, 101 (6)), serotype 5 (16.7%), serotype 4 (8.5%) and serotype 6 : 1013-1019).

Thus, the present inventors have made intensive efforts to develop a vector for the production of serotype 6 recombinant adenovirus. As a result, when a shuttle vector and a rescue vector containing genetic information of serotype 6 recombinant adenovirus are prepared and homologous recombination is performed in E. coli, 6 recombinant adenovirus can be produced, and the present invention has been completed.

It is an object of the present invention to provide a shuttle vector and a rescue vector for producing a vector for the production of serotype 6 recombinant adenovirus.

Another object of the present invention is to provide a recombinant serotype 6 adenovirus vector by homologous recombination of a shuttle vector and a rescue vector, and a recombinant serotype 6 adenovirus vector produced by the method.

It is still another object of the present invention to provide a serotype recombinant adenovirus vector for expressing a target protein in which a gene encoding a target protein is introduced into the serotype 6 recombinant adenovirus vector.

In order to accomplish the above object, the present invention provides a recombinant vector comprising a left ad 6 inverted terminal repeat (LITR), an encapsidation signal of a serotype 6 adenovirus genome, a right ad 6 inverted terminal repeat (RITR) The present invention provides a shuttle vector for serotype 6 recombinant adenovirus, which contains the arm homology and adjacent left arm homology, does not include the E1 region of the serotype 6 adenovirus genome, and contains a replication origin and an antibiotic resistance gene.

The present invention also provides a recombinant adenoviral vector for producing serotype 6 recombinant adenovirus comprising the serotype 6 adenovirus genome from which the E1 region has been deleted, the right arm homology, and the left arm homology, the origin of replication and the antibiotic resistance gene adjacent to the right arm homology and a rescue vector.

(A) simultaneously transforming the shuttle vector and the rescue vector into a microorganism having a recombinase function; (b) culturing the transformed microorganism; And (c) obtaining a recombinant serotype 6 adenovirus vector from the cultured microorganism.

The present invention also provides a recombinant serotype 6 adenovirus vector which comprises the serotype 6 adenovirus genome, the promoter, the origin of replication and the antibiotic resistance gene, which is produced by the above method and does not contain the E1 gene.

The present invention also provides a method of producing a recombinant serotype 6 adenovirus vector, characterized in that the recombinant serotype 6 adenovirus vector is infected into a cell line expressing the E1 protein and the recombinant serotype 6 adenovirus is obtained from the infected cell line ≪ / RTI >

The present invention also provides a method for producing a target protein, wherein the recombinant cell line is cultured to express a target protein, and then the expressed target protein is obtained.

The present invention also provides a gene therapy agent, a cell gene therapy agent and a vaccine containing the serotype 6 recombinant adenovirus vector.

According to the present invention, it is possible to easily and efficiently produce serotype 6 adenovirus having a low probability of an immune response, which is useful for producing gene therapy agents, cell gene therapy agents and vaccines.

Figure 1 is a genomic map of wild-type serotype 6 adenovirus.
Figure 2 shows the vectors used in the construction of pAd6-Transfer and pAd6-Simple (A: pGEM-11Zf (+) vector used in the production of rAd6; B: pGEM-11Zf Results of base sequence analysis).
FIG. 3 shows the production process of pGEM-11Zf (+) - YC2. (A: Wild type Ad6 has 1 to 558 PCR amplified regions, and forward primer and reverse primer have Pac I and Afl II flanking sequences. . B: the annealing temperature to Gradient PCR was set at 55 ℃ C: pGEM-11Zf ( +) - Pac I, Afl II cut electrophoresis confirmed the results of YC1; D: pGEM-11Zf ( +) - plasmid YC2 map ).
Figure 4 shows the production process of pGEM-11Zf (+) - YC3 (A: Afl II to Xho I cleavage sites after the E1 region as part of the wild type Ad6 map.) B and C: Multiple clones were amplified with Afl II and Bam HI (B) and Xho I (C). D: Plasmid map of pGEM-11Zf (+) - YC3).
Figure 5 shows the procedure for the construction of pGEM-11Zf (+) - YC3-AsisSI (A: PCR primer to add Asi SI to pGEM-11Zf (+) - YC3; B: 5 out of 5 clones has an insert size of the C:. - pGEM-11Zf ( +) sequences of the D Asi SI to YC3:. pGEM-11Zf (+ ) - map of the plasmid genes YC3-AsiSI M: 100 bp DNA ladder)
Figure 6 shows the production process of pGEM-11Zf (+) - YC3-AsisSI-AsisSI (A: PCR method for adding Asi SI to the RITR flanking region, B: gradient PCR for annealing temperature setting, Plasmid map of D: pGEM-11Zf (+) - YC3-AsisSI-AsisSI), obtained by sequencing three clones (4-2. 4-7, 5-9).
7: Screening results of pGEM-11Zf (+) - YC4-MCS1 (A: Screening results of pGEM-11Zf (+) - YC4-MCS1; B: Clone No. 1 as Xba I, Xho I, Sal I , the result is cut by Afl II, make a single cut; C: pGEM-11Zf (+ ) - a plasmid map of YC4-MCS1).
(A) pGEM-11Zf (+) - YC4-MCS1 vector B. The backbone of pShuttle-Ad5 with the Pac I cleavage site (Kana, Ori) was substituted with pGEM-11Zf +) - YC4-MCS1; C: map of pAd6-Transfer vector.
FIG. 9 shows the production process of pGEM-11Zf (+) - YC2-R1 (A: Plasmid map of pGEM-11Zf (+) - YC2; B: Plasmid map of pGEM-11Zf (+) - YC2-R1; C: Result of nucleotide sequence analysis)
FIG. 10 shows the production process of YC2-R1-1 (A. pGEM-11Zf (+) - YC2-R1 in which two Pac I cleavage sites and PCR primers are located; Screening results: C: part of the result of nucleotide sequence analysis for clone 1; D: plasmid map of YC2-R1-1).
Figure 11 shows the production process of YC2-R2 (A. Genomic map of serotype 6 Ad6. Afl II (3,530 nt) and Bst BI site were cloned into Afl II, Bst BI of YC2-R1-1. B: EcoRV cleavage result; C: plasmid map of YC2-R2).
Figure 12 shows the production process of YC2-R3 (screening of A. YC2-R3; B: YC2-R3, clone 21 was further digested with Eco RV or Hind III; Plasmid map).
Fig. 13 shows a process of removing 1 to 558 nt of adenovirus from YC2-R3 (A: YC2-R3 plasmid map, B: PCR primer position using YC2-R3 as a template).
Figure 14 illustrates a manufacturing process of YC2-R3-2 (A: clone screening (Cla I & Afl II digestion) ; B: clone confirmed (Eco RI digestion); C: Plasmid map of YC2-R3-2).
B: Gradient PCR (stomach: 11.8 Kb PCR product, below: 3.5 Kb PCR product), C: YC2-R4 (SEQ ID NO: -2 clone ( Hind III cut), D: YC2-R4-2 plasmid map).
FIG. 16 shows the PCR process for the production of pAd6-Simple2 (A: gene map and PCR amplification region of wild-type serotype 6 adenovirus;
FIG. 17 shows the plasmid map (A) and the restriction enzyme treatment result (B) of pAd6-Simple2.
Figure 18 shows the production process of the pAd6-Transfer-M3-GFP vector (A. pAd6-Transfer vector plasmid map B: pAAV-M3-GFP plasmid map C: pAd6-Transfer-M3-GFP vector Plasmid map; D: pAd6-Transfer-M3-GFP treated with Bgl II (expected size: 7,117 bp & 1,348 bp)).
19 is a schematic diagram showing a process of homologous recombination of Ad6-Transfer vector and Ad6-Simple2 vector in Escherichia coli (BJ5183).
Figure 20 is a map of the recombinant pAd6-M3-GFP vector generated by homologous recombination.
FIG. 21 is a photograph of the recombinant pAd6-M3-GFP vector fragmented with restriction enzymes to select it, followed by electrophoresis. A is the treated DNA with Pac1 restriction enzyme. The clones that meet the expected results are clones of lane 1, lane 4, lane 6, lane 10. B is a picture obtained by re-transforming clone of lane 6 into TOP-10 cells and then digesting with restriction enzyme by electrophoresis. The left side shows the digestion with PacI, BstBI and HindIII restriction enzymes, and the right side shows the digest with AsiSI restriction enzyme.

In one aspect, the present invention is directed to a method for the detection and / or identification of a human adenoviral genome comprising the steps of: (a) constructing a human adenoviral vector comprising a left ad 6 inverted terminal repeat (LITR), an encapsidation signal, a right Ad6 inverted terminal repeat (RITR) A shuttle vector for serotype 6 recombinant adenovirus comprising contiguous left arm homology, which does not include the E1 region of the serotype 6 adenovirus genome, and contains a replication origin and an antibiotic resistance gene.

The shuttle vector of the present invention can be used to remove the nucleotide sequence of the modified wild-type serotype 6 adenovirus, multiple cloning sites for cloning of the desired gene, antibiotic resistance gene for clone selection, replication origin and antibiotic resistance gene And has a restriction enzyme cleavage site that can be made.

The nucleotide sequence of the modified wild-type serotype 6 adenovirus includes an encapsidation signal region that is involved in virus packaging while removing the E1 region for self-replication from the wild type sequence. It also contains a right arm homology with RITR and a left arm homology with LITR, with a unique restriction site between them.

In one embodiment of the present invention, a 702 bp pAd-transfer vector having all of the above characteristics was prepared and verified by restriction enzyme analysis.

The shuttle vector of the present invention may further contain a gene encoding a target protein, and preferably the target protein may be a therapeutic protein.

The shuttle vector of the present invention may be characterized in that the replication origin and the antibiotic resistance gene have a structure capable of being removed by a restriction enzyme.

In the present invention, the LITR (left Ad6 inverted terminal repeat) may have a sequence of 2,939 to 2,998 of the nucleotide sequence of SEQ ID NO: 1, and the right AD6 inverted terminal repeat may have the sequence of SEQ ID NO: It may be characterized by having a sequence of 6,946 to 7,005 in the nucleotide sequence.

In the present invention, the encapsidation signal of the nucleic acid may have a sequence of 3,120 to 3,269 of the nucleotide sequence of SEQ ID NO: 1, and the right arm homology may include 3,604 to 3,604 of the nucleotide sequence of SEQ ID NO: 5,848, and the left arm homology may have a sequence of 5,855 to 7,006 in the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the left arm homology and the right arm homology sequences are included both in the shuttle vector and the rescue vector used together, and homologous recombination occurs in the left arm homology and the right arm homology.

In another aspect, the present invention provides a method of producing serotype 6 recombinant adenovirus comprising the serotype 6 adenovirus genome from which the E1 region has been deleted, the right arm homology and the right arm homology and the adjacent arm homology, origin of replication and antibiotic resistance gene Gt; rescue < / RTI >

In one embodiment of the present invention, the rescue vector has all of the nucleotide sequences of the serotype 6 adenovirus from which the E1 region has been removed, and removes the LITR to AflII (1 to 3,530 nt) region of the serotype 6 adenovirus, A pAd-Simple2 vector containing the serotype 6 adenovirus AflII (3,530 nt) to the right ITR (35,759 nt) was constructed.

The rescue vector of the present invention may be characterized in that the E1 region deleted from the serotype 6 adenovirus genome includes the LITR to AFIII region (GenBank No. HQ413315.1, 1 to 3,530 nt). .

In the present invention, the serotype 6 adenovirus genome from which the E1 region has been deleted may have a nucleotide sequence of 10 to 32,251 of the nucleotide sequence of SEQ ID NO: 2, and the right arm homology may include a nucleotide sequence of SEQ ID NO: And 3,604 to 5,848 of the sequence, and the left arm homology may have a sequence of 5,855 to 7,006 of the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a method for regenerating a shuttle vector comprising: (a) simultaneously transforming the shuttle vector and the rescue vector into microorganisms having a recombinase function; (b) culturing the transformed microorganism; And (c) obtaining a recombinant serotype 6 adenovirus vector from the cultured microorganism. Also provided is a method for producing a recombinant serotype 6 adenovirus vector comprising the serotype 6 An adenovirus genome, a promoter, a replication origin, and an antibiotic resistance gene. The present invention also relates to a recombinant serotype 6 adenovirus vector.

The vector for preparing serotype 6 recombinant adenovirus according to the present invention can conveniently produce serotype 6 recombinant adenovirus because it contains various restriction enzyme sites, an antibiotic resistance gene and a multiple cloning site.

The serotype 6 recombinant adenovirus produced by using the vector for the production of the serotype 6 recombinant adenovirus of the present invention can be applied to the development of a gene carrier, a gene therapy agent, a cell therapeutic agent, a vaccine and the like, and the kit type, serotype 6 recombinant adenovirus production It can be provided for research or therapeutic purposes in the form of services.

In the present invention, the shuttle vector is preferably introduced into the microorganism by linearizing the right arm homology and the left arm homology linkage sites with restriction enzymes.

In one embodiment of the present invention, the recombinant viral vector for serotype 6 recombinant adenovirus production comprises two types of plasmid vectors, pAd6-Transfer (shuttle vector) and pAd6-Simple2 (rescue vector).

The shuttle vector and the rescue vector of the present invention can be transformed into bacteria having a recombinant enzyme function to produce serotype 6 recombinant adenovirus by homologous recombination.

In one embodiment of the present invention, pAd6-Transfer-M3-GFP is prepared by inserting a gene coding for GFP using GFP as a target protein in the pAd6-Transfer shuttle vector, and then pAd6-Simple2 and Escherichia coli (BJ5183) The homologous recombination was induced, and the cell line expressing GFP was successfully screened to confirm that the vector for serotype 6 recombinant adenovirus production of the present invention was able to successfully produce serotype 6 recombinant adenovirus.

In the present invention, the microorganism having recombinase activity may be selected from the group consisting of E. coli, EL250 cells, EL350 cells and adenovirus-producing cell lines.

The recombinant serotype 6 adenovirus vector of the present invention may include a serotype 6 adenovirus genome that does not contain the E1 gene, a promoter, a replication origin, and an antibiotic resistance gene.

The recombinant serotype 6 adenovirus vector of the present invention preferably further comprises a gene encoding a target protein, and the target protein may be a therapeutic protein.

As used herein, the term " vector " means a DNA product containing a DNA sequence operably linked to an appropriate regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " plasmid " and " vector " are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or known in the art. Typical expression vectors for mammalian cell culture expression are based on, for example, pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen).

The expression " expression control sequence " means a DNA sequence that is essential for the expression of a coding sequence operably linked to a particular host organism. Such regulatory sequences include promoters for carrying out transcription, any operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. For example, regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites.

A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way that the appropriate molecule (e. G., Transcriptional activator protein) is capable of gene expression when bound to the regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, " operably linked " means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As is well known in the art, to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to a transcriptional and detoxification regulatory sequence that functions in the selected expression host. Preferably the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector should further comprise a useful expression marker in the eukaryotic expression host.

As used herein, the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration. As used herein, the term " transfection " means that an expression vector, whether or not any coding sequence is actually expressed, is accepted by the host cell.

The host cell of the invention may be a prokaryotic or eukaryotic cell. In addition, a host having high efficiency of introduction of DNA and high efficiency of expression of the introduced DNA is usually used. this. Known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, and yeast, insect cells such as Spodoptera prougiperata (SF9), animal cells such as CHO and mouse cells, COS 1, COS 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used.

In order to homologously recombine the shuttle vector and the rescue vector of the present invention, it is preferable to transform the two vectors into a host cell containing recombinase.

In another aspect, the present invention provides a recombinant cell line having the ability to express a target protein, wherein the recombinant serotype 6 adenovirus vector is introduced into a cell line expressing the E1 protein, and the recombinant cell line, and the recombinant cell line is cultured from the cultured recombinant cell line Type 6 adenovirus vector, wherein the recombinant serotype 6 adenovirus vector is obtained by cultivating the recombinant serotype 6 adenovirus vector.

In another aspect, the present invention relates to a method for producing a target protein, which comprises culturing the recombinant cell line to express a target protein, and then obtaining the expressed target protein.

In another aspect, the present invention relates to a gene therapy agent containing the serotype 6 recombinant adenovirus vector.

In addition, the vector of the present invention can be modified, modified, and applied in the field of genetic engineering.

In the present invention, the gene therapy agent includes a cell gene therapy agent using cells infected with the serotype 6 recombinant adenovirus vector, and a vaccine containing the cells infected with the serotype 6 recombinant adenovirus vector or the serotype 6 recombinant adenovirus vector.

The vector of the present invention can be used for the purpose of prevention and treatment of various diseases in the medical and veterinary fields by inserting a nucleic acid encoding one or more genes to be transferred and expressed into cells, tissues and organs into a vector.

The gene that may be inserted into the vector of the present invention may be a hormone, a cytokine, an enzyme, an antibody, a growth factor, a transcription regulator, a blood factor, a vaccine, a structural protein, a drug susceptibility gene, a drug resistance gene, a ligand protein and a receptor, But are not limited to, genes encoding various physiologically active peptides such as antigens and receptor antagonists, derivatives thereof, and the like. In addition, antisense nucleic acids that regulate gene expression in target cells or nucleic acids that express siRNA can be included.

The serotype 6 recombinant adenovirus may be produced by ex vivo method of transferring cells to a subject isolated from the subject, and in vivo method of directly injecting the subject into a subject It can be used not only as a human but also as an animal carrier for an animal having an ability to infect by a virus.

The serotype 6 recombinant adenovirus of the present invention is useful for the treatment and prevention of diseases such as hematological diseases such as hemophilia and orthopedic anemia, diseases of the central nervous system such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease and genetic defects, diabetes, growth hormone deficiency and osteoporosis Autoimmune diseases such as hyperlipidemia, multiple sclerosis, psoriasis and rheumatic diseases, HIV infection, infectious diseases such as influenza, papillomavirus and mycobacteria, and tumors.

The serotype 6 recombinant adenovirus of the present invention may be formulated into an appropriate formulation together with an acceptable pharmaceutical carrier and administered in a variety of routes, for example, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, , Topical administration, intranasal administration, intrapulmonary administration, rectal administration.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Example 1 Genomic Gene Isolation of Serotype 6 Adenovirus (Ad6)

Wild-type serotype 6 adenovirus (Tonsil 99; ATCC VR-6, USA) was propagated in A549 (ATCC CLL 185) cells as follows. A549 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% FBS, 1% Penicillin (100 U / ml) and streptomycin (100 mg / ml) at 37 ° C in a 5% CO2 incubator.

Wild-type Ad6 was infected with about 1 TCID ₅₀ / cell into 100 10-cm dish A549 cells seeded at about 70% confluency and harvested at 3 days. After three freeze / thaw cycles, the cell debris-free supernatant was separated by CsCl-gradient ultra-centrifugation and used for genomic DNA isolation of wild-type Ad6. 350 μl of the fraction containing the wild-type Ad6 genome separated by CsCl-gradient ultra-centrifugation method was reacted with benzonase (30 Units, Novagen) at 37 ° C for 1 hour and then 10 μl of 0.5 M EDTA (pH 8.0) K (10 mg / ml), 20 μl of 10% SDS, and reacted overnight at 55 ° C.

After phenol / chloroform extraction, Ad6 DNA was isolated by ethanol precipitation. The wild-type serotype 6 adenovirus has a unique restriction site of ClaI (918 nt), BstBI (10,674 nt), PmeI (13,251 nt), PacI (28,602 nt) and SwaI (28,887 nt).

Example 2: Construction of a shuttle vector (pAd6-Transfer)

PAd6-Transfer, which is required for serotype 6 recombinant adenovirus, has right arm homology (including RITR) and left arm homology (including LITR) of serotype 6 adenoviruses without the E1 region and between right arm homology and right arm homology There should be unique restriction enzyme cleavage sites, multiple cloning sites for the cloning of desired genes, kanamycin resistance gene for easy clone selection, and a restriction enzyme for recombinant adenovirus production through transfection Should be able to remove the kanamycin resistance gene.

The pAd6-Transfer was prepared in seven steps. Ad6-Transfer can produce a recombinant adenovirus lacking its own replication ability by removing the E1 region (559-3,505 nt) of the wild-type serotype 6 adenovirus to a size of 7,022 bp. It has multiple cloning sites to clone a variety of DNA. It has a left arm homology and a right arm homology required for homologous recombination with pAd-Simple2, and has a unique XhoI restriction site between the two. The characteristics and positions of the specific base sequences present in the pAd6-Transfer are shown in Table 1.

Characteristics and nucleotide position of pAd6-Transfer vector Feature Nucleotide Position in pAd6-Transfer Genebank No. Nucleotide Position in adenovirus 6 Left Ad6 inverted terminal repeat (LITR) 2,939-2,998 HQ413315.1 1 to 102 Encapsidation signal 3,120 ~ 3,269 HQ413315.1 182 to 331 Multiple cloning site 3,498 to 3,600 N / A N / A Right Ad6 inverted terminal repeat (RITR) 6,946 to 7,005 HQ413315.1 35,657 to 35,759 pBR322 origin of replication 128 ~ 906 N / A N / A Kanamycin resistance ORF 1,664 ~ 2,466 N / A N / A Left arm homology 5,855-7,006 HQ413315.1 34,608 to 35,759 Right arm homology 3,604 to 5,848 HQ413315.1 3,535 ~ 5,779

The vector pAd6-Transfer was constructed through the following seven steps (Table 2).

Production steps of pAd6-Transfer Production process Contents Step 1 Construction of pGEM-11Zf (+) - YC1 with restriction enzyme cleavage site for cloning Step 2 Construction of pGEM-11Zf (+) - YC2 by cloning region (1 ~ 558 nt) containing LITR of serotype 6 adenovirus into pGEM-11Zf Step 3 Construction of pGEM-11Zf (+) - YC3 by cloning regions containing AflII (3530 nt) to XhoI (5,781 nt) of serotype 6 adenovirus into pGEM-11Zf Step 4 Construction of pGEM-11Zf (+) - YC3-AsiSI by inserting one AsiSI cleavage site into pGEM-11Zf (+) - YC3 Step 5 Construction of pGEM-11Zf (+) - YC3-AsiSI-AsiSI by inserting AsiSI cleavage site into pGEM-11Zf (+) - YC3-AsiSI Step 6 Multiple cloning sites were inserted into pGEM-11Zf (+) - YC3-AsiSI-AsiSI
Production of pGEM-11Zf (+) - YC4-MCS1 Step 7 The pAd6-Transfer vector was constructed by replacing the ampicillin resistance gene of pGEM-11Zf (+) - YC4-MCS1 with the Kanamycine resistance gene

Two types of vectors, pAd6-Transfer and pAd6-Simple2, are used for the production of serotype recombinant adenovirus (rAd6). For the construction of pAd6-Simple2, about 37 Kb or more should be cloned. The pGEM-11Zf (+) vector, which has been reported to be cloned up to 100 Kb, was used in the construction of pAd6-Transfer and pAd6-Simple2.

(1) Production of pGEM-11Zf (+) - YCl

The pGEM-11Zf (+) vector (Promega, USA) was purchased and used to construct pGEM-11Zf (+) - YC1 with restriction enzyme cleavage sites necessary for cloning of serotype 6 recombinant adenovirus. A plasmid map of the pGEM-11Zf (+) vector is shown in Fig. 2A. Various enzymes were purchased from Roche (Germany), Fermentas (Thermo, USA) or NEB (USA). The wild-type serotype 6 recombinant adenovirus is unique in ClaI, BstBI, PacI, PmeI, SwaI restriction sites and should be used to remove the E1 region (E1a, E1b 19K, E1b 55K) do. The pGEM-11Zf (+) digested with Sfi I and Hind III synthetic gene Sfi I - Pac I - Afl II - Pme I - Eco RV - Swa I - Sna BI - Xho I - Bam HI - Hind III (5'- GGC CAA GTC GGC CTT AAT TAA CTT AAG GTT TAA ACG ATA TCA TTT AAA TTA CGT ACT CGA GGG ATC CAA GCT T-3 '(SEQ ID NO: 3) was cloned (Bioneer, Korea) +) - YC1 (Fig. 2B). The cloned region in pGEM-11Zf (+) - YCl was confirmed by sequencing (FIG. 2C).

(2) Production of pGEM-11Zf (+) - YC2

pGEM-11Zf (+) - YC2 was prepared by cloning a region (1 to 558 nt) containing LITR of serotype 6 adenovirus into pGEM-11Zf (+) - YC1.

Approximately 559 to 3505 nucleotides (nt) in the wild-type Ad6 genome encode the E1 region (E1a, E1b 19K, E1b 55K). The E1 region is required for the function of the adenovirus self-replication, and the recombinant adenovirus lacking the replication ability must remove the E1 region. Instead, the E1 protein is provided in HEK293 cells or rAd producing cell lines such as PER.C6. 11Zf (+) - YC1 prepared in the above step was firstly PCR amplified from 1 to 558 nucleotides of the wild-type serotype 6 adenovirus in order to remove only the E1 region, and the vector was cloned into pGEM-11Zf -YC2. To facilitate the cloning of the 1 to 558 nucleotides region of Ad6, the Pac I cleavage site and the Afl II cleavage site were flanked on the forward primer (FIG. 3A).

Forward primer: 5'-ATG CTT AAT TAA ATC ATC AAT AAT ATA CCT TAT TTT GGA TTG-3 '(SEQ ID NO: 4)

Reverse primer: 5'-ATG CCT TAA GTT TCA GTC CCG GTG TCG GAG CGG CTC GGA G-3 (SEQ ID NO: 5)

As a result of gradient PCR with serotype 6 adenovirus as a template, it was confirmed that the optimum temperature was 55 ° C (FIG. 3B), and the DNA polymerization reaction was carried out with Taq polymerase (Bioprince, China) . The amplified DNA product was isolated and cloned into pGEM-11Zf (+) - YC2 digested with Pac I and Afl II. Four clones having 558 bp among several clones were selected (Fig. 3C). A plasmid map of the prepared pGEM-11Zf (+) - YC2 is shown in FIG. 3D.

(3) Production of pGEM-11Zf (+) - YC3

pGEM-11Zf (+) - YC3 was prepared by cloning 3,530 ( Afl II) to 5,781 ( Xho I) regions (FIG. 4A) of wild type serotype 6 adenovirus into pGEM-11Zf (+) - YC2. Ad6 DNA isolated by CsCl-gradient ultra-centrifugation was treated with restriction enzymes Afl II and Xho I and cloned into pGEM-11Zf (+) - YC2 cut with Afl II and Xho I. Seven clones were firstly screened (Fig. 4B) by restriction enzyme analysis and confirmed with other restriction enzymes (Fig. 4C). One clone of seven clones was used for the construction of pGEM-11Zf (+) - YC3. A plasmid map of the prepared pGEM-11Zf (+) - YC3 is shown in Fig. 4D.

(4) Preparation of pGEM-11Zf (+) - YC3-AsiSI

One AsiSI cleavage site was inserted into pGEM-11Zf (+) - YC3 to construct pGEM-11Zf (+) - YC3-AsiSI (FIG.

In order to produce recombinant adenovirus using two kinds of plasmid vectors, competent cells having recombinase should be transformed. The recombinant plasmid thus constructed has a kanamycin resistance gene, and two restriction enzyme cleavage sites capable of removing the backbone containing the kanamycin resistance gene before transfection into the adenovirus producing cell line should be provided.

First, the AsisSI cleavage site was inserted into the flanking region of Ad5 adenovirus 1 to 558 nt. Pac I and Asi SI flanking sequences were used as forward primer and reverse primer corresponding to 667 to 690 nt sites of pGEM-11Zf (+) - YC3 with Pac I treated pGEM-11Zf (+) - .

forward primer: 5'-ATT TAA TTA AGC GAT CGC ATC ATC AAT AAT ATA CCT TAT TTT GGA TTG-3 '(SEQ ID NO: 6)

reverse primer: 5'-ATG CTT CCA TCA AAC GAG TTG GCG-3 '(SEQ ID NO: 7)

PCR products and pGEM-11Zf (+) - YC3 are Pac I and Afl II and then digested with, each other ligated to a single clone (pGEM-11Zf (+) - YC3-AsiSI) have gained, after sequencing pGEM- 11Zf (+) - YC3-AsiSI-AsiSI.

(5) Construction of pGEM-11Zf (+) - YC3-AsiSI-AsiSI

 AsiSI cleavage site was inserted into pGEM-11Zf (+) - YC3-AsiSI to construct pGEM-11Zf (+) - YC3-AsiSI-AsiSI (FIG.

The RITR region of the cloned serotype 6 adenovirus was amplified using a forward primer with Pac I and Asi SI cleavage sites and a reverse primer corresponding to the vector sequence.

forward primer: 5'-TCT TAC CTC TGG TTT CCA TGA GCC-3 '(SEQ ID NO: 8)

reverse primer: 5'-att taa tta agc gat cgc CAT CAT CAA TAA TAT ACC TTA TTT TGG-3 '(SEQ ID NO: 9)

The annealing temperature was 53 ° C and the following procedure was carried out using clones 5-9 whose nucleotide sequences corresponded to those of the three clones (4-2, 4-7, 5-9).

(6) Production of pGEM-11Zf (+) - YC4-MCS1

Multiple cloning sites were inserted into pGEM-11Zf (+) - YC3-AsiSI-AsiSI to construct pGEM-11Zf (+) - YC4-MCS1 (FIG.

A multiple cloning site for inserting the desired gene pGEM-11Zf (+) - to insert a YC3-AsiSI-AsiSI, pGEM- T vector to Afl II- Sal I- Cla I- Pme I- Not I- Eco RV - Eco RI- Swa I- Nhe I- Bgl II- Bst BI- Sna BI- was cloned into the Afl II after synthesis (Bioneer, Korea), and then digested with Afl II-cut the multiple cloning sites into Afl II pGEM-11Zf ( +) - YC3-AsiSI-AsiSI. ≪ / RTI > Clones were screened for Asi SI and Pme I, and the next step was performed using clone No. 1 with multiple cloning sites.

(7) Construction of pAd6-Transfer vector

The pAd6-Transfer vector was prepared by replacing the ampicillin resistance gene of pGEM-11Zf (+) - YC4-MCS1 with the Kanamycine resistance gene (Fig. 8).

The backbone containing the ampicillin resistance gene was removed from pGEM-11Zf (+) - YC4-MCS1 with the ampicillin resistance gene and a kanamycin resistance gene was inserted. pGEM-11Zf (+) - in YC4-MCS1 ampicillin is cutting the kanamycin resistance gene of removing the backbone, including the ampicillin resistance gene cleaved with Pac I, and present in the backbone of the pShuttle-Ad5 (Qbiogen, USA) with Pac I After ligation, pAd6-Transfer vector was constructed.

Example 3: Construction of rescue vector (pAD5-Simple2)

PAd6-Simple2, which is required for serotype 6 recombinant adenovirus production, should have only the E1 region removed and all plasmid 6 adenovirus (Ad6) in plasmid form. Serotype 6 adenoviruses are 35,759 bp in size, making it difficult to clone all of the genes into plasmids at once. Namely, pAd6-Simple2 was prepared by sequential cloning into a vector having a unique restriction enzyme site in serotype 6 adenovirus. pAd6-Simple2 was constructed by removing LITR to AflII (1 to 3,530 nt) containing the E1 region of the serotype 6 adenovirus. Recombinant pAd6-Simple2 contains the wild-type serotype 6 adenovirus AflII (3,530 nt) to the right ITR (7,158 nt), lacking the E1 region. The characteristics and positions of the specific nucleotide sequences present in the pAd6-Transfer are shown in Table 3.

Characteristics and nucleotide position of pAd6-Simple2 vector Feature Nucleotide Position in pAd6-Simple2 Genebank No. Nucleotide Position in adenovirus 6 f1 origin of replication 34,764 ~ 35,219 N / A N / A ampicillin resistance ORF 33,467 to 34,327 N / A N / A Serotype 6 Adenovirus without E1 10 to 32,251 HQ413315.1 3,529 to 35,759 Right arm homology 16-2260 HQ413315.1 3,535 ~ 5,779 Left arm homology 31,089 to 32,240 HQ413315.1 34,608 to 35,759 Origin of replication 32,666 to 33,221 N / A N / A

The rescue vector (pAD5-Simple2) was constructed through the following seven steps (Table 4).

Production steps of pAD5-Simple2 Step 1 pGEM-11Zf (+) - YC2 containing restriction enzymes Afl II, Bst BI, Pme I, Pac I and Cla I
Construction of pGEM-11Zf (+) - YC2-R1 Step 2 One of the two Pac I sites present in pGEM-11Zf (+) - YC2-R1 was removed to produce YC2-R1-1 Step 3 Production of YC2-R2 by inserting Afl II (3,530 nt) to Bst BI (10,674 nt) of serotype 6 adenovirus into YC2-R1-1 Step 4 The production of YC2-R3 by inserting Bst BI (10,674 nt) to Pme I (13,251 nt) of serotype 6 adenovirus into YC2-R2 Step 5 In YC2-R3, 1 ~ 558 nt of serotype 6 adenovirus was removed to produce YC2-R3-2 Step 6 Production of YC2-R4-2 by inserting Pme I (9,736 nt) to Pac I (25,077 nt) in YC2-R3-2 Step 7 The production of pAd6-Simple2 by inserting Pac I (25,086 nt) to RITR end (32,251 nt) of serotype 6 adenovirus into YC2-R4-2

(1) Production of pGEM-11Zf (+) - YC2-R1

pGEM-11Zf (+) - YC2-R1 containing restriction enzymes Afl II, Bst BI, Pme I, Pac I and Cla I was prepared in pGEM-11Zf (+) - YC2 (FIG.

Multiple cloning sites ( Afl II- Sac I- Bst BI- Xho I- Pme I- Eco RV- Pac I- Bam HI- Swa I- Xba I- Cla I - HindIII ; 5'-CTT AAG GAG CTC TTC GAA CTC GAG GTT TAA ACG ATA TCT TAA TTA AGG ATC CAT TTA AAT TCT AGA ATC GAT AAG CTT-3 ': SEQ ID NO: 10) (Bioneer, Korea), and the multiple cloning site (MCS) was confirmed by sequencing. The pGEM-11Zf (+) - YC2-

(2) Production of YC2-R1-1

One of the two PacI sites existing in pGEM-11Zf (+) - YC2-R1 was removed to prepare YC2-R1-1 (FIG. 10).

In the final step (Step 7) of pAd6-Simple2 production, Pac I is used, and there are two Pac I cleavage sites in pGEM-11Zf (+) - YC2-R1. To facilitate pAd6-Simple2 cloning, the Pac I (1) cleavage site on the 5 'side of pGEM-11Zf (+) - YC2-R1 was removed. The vector pGEM-11Zf (+) - YC2-R1 was cleaved by Pac I, blunt-ended with klenow, and then digested with Hind III. Insert corresponds to 747 to 772 bp with forward primer (5'-ATC ATC AAT AAT ATA CCT TAT TTT GG-3 ': SEQ ID NO: 11) corresponding to 31-56 bp of pGEM-11Zf (5'-TCC GGC TCG TAT GTT GTG TGG AAT TG-3 ': SEQ ID NO: 12). The PCR products were blunted with klenow and then digested with Hind III. The 5 'smooth end and 3' HindIII truncated vector and insert were ligated and one of the 10 clones was selected and sequenced and used for YC2-R2 construction.

(3) Production of YC2-R2

YC2-R2 was prepared by inserting Afl II (3,530 nt) to Bst BI (10,674 nt) of serotype 6 adenovirus into YC2-R1-1 (FIG. 11).

In serotype 6 adenovirus, four Afl II cleavage sites and one Bst BI cleavage site were found, and a part of Afl II (3,530 nt) and Bst BI (10,674 nt) DNA fragments were first cloned into YC2-R1. YC2-R1-1 and serotype 6 adenovirus DNA were cut into Afl II and Bst BI, and 10 clones were screened to obtain 9 clones. When digested with EcoRV, 9426 bp, 1238 bp and 267 bp were found.

(4) Production of YC2-R3

YC2-R3 was prepared by inserting Bst BI (10,674 nt) to Pme I (13,251 nt) of serotype 6 adenovirus into YC2-R2 (FIG. 12).

Pst I (13,251 nt) region was inserted from Bst BI (10,674 nt) of the serotype 6 adenovirus genome at the BstB I and PmeI cleavage sites of YC2-R2. A total of 30 clones were screened and cleaved with Bam HI to yield 10 clones with the desired size fragments (10,905 bp, 2,587 bp). When clone # 21 was selected and digested with Eco RV and HindIII , a DNA fragment of the desired size was identified and named YC2-R3 and used in the construction of YC2-R4.

(5) Production of YC2-R3-2

YC2-R3-2 was prepared by removing 1 to 558 nt of serotype 6 adenovirus from YC2-R3.

YC2-R3 has 1 to 558 nt of serotype 6 adenovirus. Since this region has an unwanted recombination in homologous recombination in BJ5183 or may interfere with homologous recombination, the 1 to 558 nt region of serotype 6 adenovirus was removed from pYC2-R3 (Figure 13).

YC2-R3 was cut into Afl II and Cla I and used as a cloning vector. The backbone containing the Amp ^R gene was amplified by PCR with YC2-R3 and then cut into Afl II and Cla I and used as an insert. The base sequence of the primer used in the PCR was YC2-R3-ClaI (5'-atg cct taa gaa ttc (SEQ ID NO: 13) gcc cta tag tga gtc gta t-3 ': SEQ ID NO: 14).

10 clones were cut into Afl II & Cla I or Eco RI by ligation of YC2-R3 ( Afl II and Cla I digested) and PCR product ( Afl II and Cla I digested). As a result, three clones (Clone # 1, # 3, # 6) (FIG. 14).

(6) Production of YC2-R4-2

YC2-R4-2 was prepared by inserting Pme I (13,251 nt) to Pac I (28,602 nt) in YC2-R3-2 (FIG. 15).

To prepare YC2-R4-2, YC2-R3-2 was digested with Pme I and Pac I and used as a cloning vector. The region corresponding to about 15 Kb of the serotype 6 was PCR amplified by insertions ( 11.8 Kb, 3.5 Kb), and YC2-R4-2 was cloned by in-fusion reaction.

(7) Production of pAd6-Simple2

PAd6-Simple2 was constructed by inserting Pac I (25,086 nt) to RITR end (32,251 nt) of serotype 6 adenovirus into YC2-R4-2.

YC2-R4-2 was digested with Pac I and Cla I and used as a cloning vector. The RITR region (25,086 nt to 32,251 nt) was cloned in the Pac I amplified with the serotype 6 adenovirus genome as a template by in-fusion system Respectively. The nucleotide sequence of the primers used was Infu-F5 (5'-ctg gga ttt ttt aat ta gta tat gag-3 ': SEQ ID NO: 15), Infu-R7 (5'-TAC TCA AGC TTA TCG CAT CAT CAA TAA TAT ACC TTA TTT TG-3 ': SEQ ID NO: 16). FIG. 16 shows amplified regions in serotype 6, and FIG. 16B shows the results when gradient PCR was performed using primers, Infu-F5 and Infu-R7. The obtained clone was digested with Xho I or Cla I to confirm the desired size ( Xho I cuts: 14.2 kb, 11.4 kb, 5.8 kb, 2.5 kb, 1.4 kb, Cla I cuts: 35 kb) and the plasmid of pAd6- The map is shown in Fig.

Example 4 Production of Recombinant Plasmid Vector

(1) Construction of pAd6-Transfer-M3-eGFP vector

Two types of vectors were used to demonstrate that recombinant plasmid vectors were produced. In order to facilitate gene expression analysis, a cassette capable of expressing eGFP was cloned into multiple cloning sites of pAd6-Transfer vector to prepare pAd6-Transfer-M3-eGFP.

The M3 promoter is known to be a cancer-specific promoter. A pAd6-Transfer-M3-GFP vector was constructed to express the reporter gene GFP under the control of the M3 promoter. The pAd6-Transfer vector was treated with Pme I and Not I restriction enzymes, and the pAAV-M3-GFP vector (Korean Patent No. 1,204,895) was treated with restriction enzymes Sma I and Not I to separate the M3 promoter and the GFP DNA fragment Pme I and Sma I are blunt end). The vector and the insert were ligated with each other to construct a pAd6-Transfer-M3-GFP vector (Fig. 18).

(2) homologous recombination of vectors

The above pAd6-Transfer-M3-eGFP vector has one XhoI restriction enzyme site. For homologous recombination with pAd6-Simple2, pAd6-Transfer-M3-eGFP was treated with XhoI to linearize the structure. To induce homologous recombination, pAd6-Transfer-M3-eGFP linearized in E. coli (BJ5183) and cyclic pAd6-Simple2 were co-transformed. A schematic diagram of a homologous recombination process for producing recombinant pAd6-Transfer-M3-eGFP is shown in Fig. The vector obtained when successful homologous recombination has restriction sites for the replication origin, antibiotic resistance gene, M3 promoter, GFP gene and PacI, BstBI, HindIII and AsiSi (Fig. 20).

(3) Screening of recombinant vector

Twelve clones with antibiotic resistance were analyzed after co-transformation with BJ5183 to select strains that were successfully recombinant. When DNA isolated from BJ5183 is treated with PacI, the expected size of the DNA fragment is 27,192 bp, 7,171 bp, and 2930 bp. Recombinant pAd6-Transfer-M3-GFP obtained from 12 clones was digested with PacI restriction enzyme and then electrophoresed to select four clones having the expected fragment size (lane 1, lane 4, lane 6, lane 10) (Fig. 21A). DNA isolated from BJ5183 was reduced in volume and resolved into TOP-10 (Invitrogen, USA), a commonly used competent cell with low resolution. As an example, DNA of clone 6 was amplified. Since the recombinant vector had restriction sites of PacI, BstBI, and HindIII, these enzymes were treated and electrophoresed to confirm the size of the sections. The expected size of DNA fragments when treated with BstBI was 28,924 bp, 7,178 bp and 1,191 bp, and the predicted sizes when treated with HindIII were 8,777, 5,324, 4,966, 3,420,3,292, 3,074, 2,388, 2,276, 2,081, 1,311 , 309, 75 bp. As a result of analysis by electrophoresis, a DNA fragment of a predicted size was observed, and a clone in which the pAd6-Transfer vector prepared in the present invention and pAd6-Simple2 were successfully recombined was selected (Fig. 21B). The selected recombinant pAd6-M3-GFP was digested with AsiSI to remove unnecessary origin of replication and kanamycin resistance genes in adenovirus packaging, and transfected into adenovirus-producing cell lines (eg, HEK293 cells) To produce recombinant adenovirus.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> SCT          Industry Academy Coopteration Groop of Chungbuk Health & Science University <120> Vectors for Prepaing Serotype 6 Recombinant Adenovirus <130> P13-B078 <160> 16 <170> Kopatentin 2.0 <210> 1 <211> 7022 <212> DNA <213> Artificial Sequence <220> <223> Total sequence of pAd6-Transfer <400> 1 catgcatgga tccatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 60 atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 120 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 180 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 240 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 300 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 360 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 420 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 480 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 540 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 600 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 660 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 720 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 780 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 840 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 900 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 960 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 1020 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 1080 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 1140 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 1200 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 1260 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 1320 attgctgcag ccatgagatt atcaaaaagg atcttcacct agatcctttt cacgtagaaa 1380 gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca 1440 agggaaaacg caagcgcaaa gagaaagcag gtagctggag gtgggcttac atggcgatag 1500 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct 1560 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tctcgccgcc aaggatctga 1620 tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg catgattgaa 1680 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 1740 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 1800 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag 1860 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 1920 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 1980 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 2040 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 2100 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 2160 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat 2220 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 2280 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 2340 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 2400 tcggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 2460 ttctgaattt tgttaaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 2520 cggcaacatc ccttataaat caaaagaata gaccgcgata gggttgagtg ttgttccagt 2580 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt 2640 ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt tgcggtcgag 2700 gtgccgtaaa gctctaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg 2760 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc 2820 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgcgcgct taatgcgccg 2880 ctacagggcg cgtccattcg ccattcagga tcgaattaat tcttaattaa gcgatcgcat 2940 catcaataat ataccttatt ttggattgaa gccaatatga taatgagggg gtggagtttg 3000 tgacgtggcg cggggcgtgg gaacggggcg ggtgacgtag tagtgtggcg gaagtgtgat 3060 gttgtaagtg tggcggaaca catgtaagcg ccggatgtgg taaaagtgac gtttttggtg 3120 tgcgccggtg tacacgggaa gtgacaattt tcgcgcggtt ttaggcggat gttgtagtaa 3180 atttgggcgt aaccaagtaa tatttggcca ttttcgcggg aaaactgaat aagaggaagt 3240 gaaatctgaa taattctgtg ttactcatag cgcgtaatat ttgtctaggg ccgcggggac 3300 tttgaccgtt tacgtggaga ctcgcccagg tgtttttctc aggtgttttc cgcgttccgg 3360 gtcaaagttg gcgttttatt attatagtca gctgacgcgc agtgtattta tacccggtga 3420 gttcctcaag aggccactct tgagtgccag cgagtagagt tttctcctcc gagccgctcc 3480 gacaccggga ctgaaactta aggtcgacat cgattctaga gtttaaacgc ggccgcgata 3540 tcgaattcag tactatttaa atgctagctt cgaaagatct tacgtaacta gtacgcgtct 3600 taaggtggga aagaatatat aaggtggggg tctcatgtag ttttgtatct gttttgcagc 3660 agccgccgcc atgagcgcca actcgtttga tggaagcatt gtgagctcat atttgacaac 3720 gcgcatgccc ccatgggccg gggtgcgtca gaatgtgatg ggctccagca ttgatggtcg 3780 ccccgtcctg cccgcaaact ctactacctt gacctacgag accgtgtctg gaacgccgtt 3840 ggagactgca gcctccgccg ccgcttcagc cgctgcagcc accgcccgcg ggattgtgac 3900 tgactttgct ttcctgagcc cgcttgcaag cagtgcagct tcccgttcat ccgcccgcga 3960 tgacaagttg acggctcttt tggcacaatt ggattctttg acccgggaac ttaatgtcgt 4020 ttctcagcag ctgttggatc tgcgccagca ggtttctgcc ctgaaggctt cctcccctcc 4080 caatgcggtt taaaacataa ataaaaacca gactctgttt ggatttggat caagcaagtg 4140 tcttgctgtc tttatttagg ggttttgcgc gcgcggtagg cccgggacca gcggtctcgg 4200 tcgttgaggg tcctgtgtat tttttccagg acgtggtaaa ggtgactctg gatgttcaga 4260 tacatgggca taagcccgtc tctggggtgg aggtagcacc actgcagagc ttcatgctgc 4320 ggggtggtgt tgtagatgat ccagtcgtag caggagcgct gggcgtggtg cctaaaaatg 4380 tctttcagta gcaagctgat tgccaggggc aggcccttgg tgtaagtgtt tacaaagcgg 4440 ttaagctggg atgggtgcat acgtggggat atgagatgca tcttggactg tatttttagg 4500 ttggctatgt tcccagccat atccctccgg ggattcatgt tgtgcagaac caccagcaca 4560 gtgtatccgg tgcacttggg aaatttgtca tgtagcttag aaggaaatgc gtggaagaac 4620 ttggagacgc ccttgtgacc tccaagattt tccatgcatt cgtccataat gatggcaatg 4680 ggcccacggg cggcggcctg ggcgaagata tttctgggat cactaacgtc atagttgtgt 4740 tccaggatga gatcgtcata ggccattttt acaaagcgcg ggcggagggt gccagactgc 4800 ggtataatgg ttccatccgg cccaggggcg tagttaccct cacagatttg catttcccac 4860 gctttgagtt cagatggggg gatcatgtct acctgcgggg cgatgaagaa aaccgtttcc 4920 ggggtagggg agatcagctg ggaagaaagc aggttcctaa gcagctgcga cttaccgcag 4980 ccggtgggcc cgtaaatcac acctattacc ggctgcaact ggtagttaag agagctgcag 5040 ctgccgtcat ccctgagcag gggggccact tcgttaagca tgtccctgac ttgcatgttt 5100 tccctgacca aatccgccag aaggcgctcg ccgcccagcg atagcagttc ttgcaaggaa 5160 gcaaagtttt tcaacggttt gaggccgtcc gccgtaggca tgcttttgag cgtttgacca 5220 agcagttcca ggcggtccca cagctcggtc acgtgctcta cggcatctcg atccagcata 5280 tctcctcgtt tcgcgggttg gggcggcttt cgctgtacgg cagtagtcgg tgctcgtcca 5340 gacgggccag ggtcatgtct ttccacgggc gcagggtcct cgtcagcgta gtctgggtca 5400 cggtgaaggg gtgcgctccg ggttgcgcgc tggccagggt gcgcttgagg ctggtcctgc 5460 tggtgctgaa gcgctgccgg tcttcgccct gcgcgtcggc caggtagcat ttgaccatgg 5520 tgtcatagtc cagcccctcc gcggcgtggc ccttggcgcg cagcttgccc ttggaggagg 5580 cgccgcacga ggggcagtgc agacttttaa gggcgtagag cttgggcgcg agaaataccg 5640 attccgggga gtaggcatcc gcgccgcagg ccccgcagac ggtctcgcat tccacgagcc 5700 aggtgagctc tggccgttcg gggtcaaaaa ccaggtttcc cccatgcttt ttgatgcgtt 5760 tcttacctct ggtttccatg agccggtgtc cacgctcggt gacgaaaagg ctgtccgtgt 5820 ccccgtatac agacttgaga ggcctgtcct cgagttcgca gggccagctg aacataatcg 5880 tgcaggtctg cacggaccag cgcggccact tccccgccag gaaccatgac aaaagaaccc 5940 acctgatta tgacacgcat actcggagct atgctaacca gcgtagcccc gatgtaagct 6000 tgttgcatgg gcggcgatat aaaatgcaag gtactgctca aaaaatcagg caaagcctcg 6060 cgcaaaaaag caagcacatc gtagtcatgc tcatgcagat aaaggcaggt aagttccgga 6120 accaccacag aaaaagacac catttttctc tcaaacatgt ctgcgggttc ctgcataaac 6180 acaaaataaa ataacaaaaa aaaaaaaaca tttaaacatt agaagcctgt cttacaacag 6240 gaaaaacaac ccttataagc ataagacgga ctacggccat gccggcgtga ccgtaaaaaa 6300 actggtcacc gtgattaaaa agcaccaccg acagttcctc ggtcatgtcc ggagtcataa 6360 tgtaagactc ggtaaacaca tcaggttggt taacatcggt cagtgctaaa aagcgaccga 6420 aatagcccgg gggaatacat acccgcaggc gtagagacaa cattacagcc cccataggag 6480 gtataacaaa attaatagga gagaaaaaca cataaacccc tgaaaaaccc tcctgcctag 6540 gcaaaatagc accctcccgc tccagaacaa catacagcgc ttccacagcg gcagccataa 6600 cagtcagcct taccagtaaa aaaacctatt aaaaaacacc actcgacacg gcaccagctc 6660 aatcagtcac agtgtaaaaa gggccaagta cagagcgagt atatatagga ctaaaaaatg 6720 acgtaacggt taaagtccac aaaaaccacc cagaaaaccg cacgcgaacc tacgcccaga 6780 aacgaaagcc aaaaaaccca caacttcctc aaatcttcac ttccgttttc ccacgatacg 6840 tcacttccca ttttaaaaaa aaactacaat tcccaataca tgcaagttac tccgccctaa 6900 aacctacgtc acccgccccg ttcccacgcc ccgcgccacg tcacaaactc caccccctca 6960 ttatcatatt ggcttcaatc caaaataagg tatattattg atgatggcga tcgcttaatt 7020 aa 7022 <210> 2 <211> 35399 <212> DNA <213> Artificial Sequence <220> <223> Total sequence of pAd6-Simple2 <400> 2 gggcgaattc ttaagggtgg gaaagaatat ataaggtggg ggtctcatgt agttttgtat 60 ctgttttgca gcagccgccg ccatgagcgc caactcgttt gatggaagca ttgtgagctc 120 atatttgaca acgcgcatgc ccccatgggc cggggtgcgt cagaatgtga tgggctccag 180 cattgatggt cgccccgtcc tgcccgcaaa ctctactacc ttgacctacg agaccgtgtc 240 tggaacgccg ttggagactg cagcctccgc cgccgcttca gccgctgcag ccaccgcccg 300 cgggattgtg actgactttg ctttcctgag cccgcttgca agcagtgcag cttcccgttc 360 atccgcccgc gatgacaagt tgacggctct tttggcacaa ttggattctt tgacccggga 420 acttaatgtc gtttctcagc agctgttgga tctgcgccag caggtttctg ccctgaaggc 480 ttcctcccct cccaatgcgg tttaaaacat aaataaaaac cagactctgt ttggatttgg 540 atcaagcaag tgtcttgctg tctttattta ggggttttgc gcgcgcggta ggcccgggac 600 cagcggtctc ggtcgttgag ggtcctgtgt attttttcca ggacgtggta aaggtgactc 660 tggatgttg gatacatggg cataagcccg tctctggggt ggaggtagca ccactgcaga 720 gcttcatgct gcggggtggt gttgtagatg atccagtcgt agcaggagcg ctgggcgtgg 780 tgcctaaaaa tgtctttcag tagcaagctg attgccaggg gcaggccctt ggtgtaagtg 840 tttacaaagc ggttaagctg ggatgggtgc atacgtgggg atatgagatg catcttggac 900 tgtattttta ggttggctat gttcccagcc atatccctcc ggggattcat gttgtgcaga 960 accaccagca cagtgtatcc ggtgcacttg ggaaatttgt catgtagctt agaaggaaat 1020 gcgtggaaga acttggagac gcccttgtga cctccaagat tttccatgca ttcgtccata 1080 atgatggcaa tgggcccacg ggcggcggcc tgggcgaaga tatttctggg atcactaacg 1140 tcatagttgt gttccaggat gagatcgtca taggccattt ttacaaagcg cgggcggagg 1200 gtgccagact gcggtataat ggttccatcc ggcccagggg cgtagttacc ctcacagatt 1260 tgcatttccc acgctttgag ttcagatggg gggatcatgt ctacctgcgg ggcgatgaag 1320 aaaaccgttt ccggggtagg ggagatcagc tgggaagaaa gcaggttcct aagcagctgc 1380 gacttaccgc agccggtggg cccgtaaatc acacctatta ccggctgcaa ctggtagtta 1440 agaggctgc agctgccgtc atccctgagc aggggggcca cttcgttaag catgtccctg 1500 acttgcatgt tttccctgac caaatccgcc agaaggcgct cgccgcccag cgatagcagt 1560 tcttgcaagg aagcaaagtt tttcaacggt ttgaggccgt ccgccgtagg catgcttttg 1620 agcgtttgac caagcagttc caggcggtcc cacagctcgg tcacgtgctc tacggcatct 1680 cgatccagca tatctcctcg tttcgcgggt tggggcggct ttcgctgtac ggcagtagtc 1740 ggtgctcgtc cagacgggcc agggtcatgt ctttccacgg gcgcagggtc ctcgtcagcg 1800 tagtctgggt cacggtgaag gggtgcgctc cgggttgcgc gctggccagg gtgcgcttga 1860 ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagc 1920 atttgaccat ggtgtcatag tccagcccct ccgcggcgtg gcccttggcg cgcagcttgc 1980 ccttggagga ggcgccgcac gaggggcagt gcagactttt aagggcgtag agcttgggcg 2040 cgagaaatac cgattccggg gagtaggcat ccgcgccgca ggccccgcag acggtctcgc 2100 attccacgag ccaggtgagc tctggccgtt cggggtcaaa aaccaggttt cccccatgct 2160 ttttgatgcg tttcttacct ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa 2220 ggctgtccgt gtccccgtat acagacttga gaggcctgtc ctcgagcggt gttccgcggt 2280 cctcctcgta tagaaactcg gaccactctg agacgaaggc tcgcgtccag gccagcacga 2340 aggaggctaa gtgggagggg tagcggtcgt tgtccactag ggggtccact cgctccaggg 2400 tgtgaagaca catgtcgccc tcttcggcat caaggaaggt gattggttta taggtgtagg 2460 ccacgtgacc gggtgttcct gaaggggggc tataaaaggg ggtgggggcg cgttcgtcct 2520 cactctcttc cgcatcgctg tctgcgaggg ccagctgttg gggtgagtac tccctctcaa 2580 aagcgggcat gacttctgcg ctaagattgt cagtttccaa aaacgaggag gatttgatat 2640 tcacctggcc cgcggtgatg cctttgaggg tggccgcgtc catctggtca gaaaagacaa 2700 tctttttgtt gtcaagcttg gtggcaaacg acccgtagag ggcgttggac agcaacttgg 2760 cgatggagcg cagggtttgg tttttgtcgc gatcggcgcg ctccttggcc gcgatgttta 2820 gctgcacgta ttcgcgcgca acgcaccgcc attcgggaaa gacggtggtg cgctcgtcgg 2880 gcactaggtg cacgcgccaa ccgcggttgt gcagggtgac aaggtcaacg ctggtggcta 2940 cctctccgcg taggcgctcg ttggtccagc agaggcggcc gcccttgcgc gagcagaatg 3000 gcggtagtgg gtctagctgc gtctcgtccg gggggtctgc gtccacggta aagaccccgg 3060 gcagcaggcg cgcgtcgaag tagtctatct tgcatccttg caagtctagc gcctgctgcc 3120 atgcgcgggc ggcaagcgcg cgctcgtatg ggttgagtgg gggaccccat ggcatggggt 3180 gggtgagcgc ggaggcgtac atgccgcaaa tgtcgtaaac gtagaggggc tctctgagta 3240 ttccaagata tgtagggtag catcttccac cgcggatgct ggcgcgcacg taatcgtata 3300 gttcgtgcga gggagcgagg aggtcgggac cgaggttgct acgggcgggc tgctctgctc 3360 ggaagactat ctgcctgaag atggcatgtg agttggatga tatggttgga cgctggaaga 3420 cgttgaagct ggcgtctgtg agacctaccg cgtcacgcac gaaggaggcg taggagtcgc 3480 gcagcttgtt gaccagctcg gcggtgacct gcacgtctag ggcgcagtag tccagggttt 3540 ccttgatgat gtcatactta tcctgtccct tttttttcca cagctcgcgg ttgaggacaa 3600 actcttcgcg gtctttccag tactcttgga tcggaaaccc gtcggcctcc gaacggtaag 3660 agcctagcat gtagaactgg ttgacggcct ggtaggcgca gcatcccttt tctacgggta 3720 gcgcgtatgc ctgcgcggcc ttccggagcg aggtgtgggt gagcgcaaag gtgtccctaa 3780 ccatgacttt gaggtactgg tatttgaagt cagtgtcgtc gcatccgccc tgctcccaga 3840 gcaaaaagtc cgtgcgcttt ttggaacgcg ggtttggcag ggcgaaggtg acatcgttga 3900 agagtatctt tcccgcgcga ggcataaagt tgcgtgtgat gcggaagggt cccggcacct 3960 cggaacggtt gttaattacc tgggcggcga gcacgatctc gtcaaagccg ttgatgttgt 4020 ggcccacaat gtaaagttcc aagaagcgcg ggatgccctt gatggaaggc aattttttaa 4080 gttcctcgta ggtgagctct tcaggggagc tgagcccgtg ctctgaaagg gcccagtctg 4140 caagatgagg gttggaagcg acgaatgagc tccacaggtc acgggccatt agcatttgca 4200 ggtggtcgcg aaaggtccta aactggcgac ctatggccat tttttctggg gtgatgcagt 4260 agaaggtaag cgggtcttgt tcccagcggt cccatccaag gtccgcggct aggtctcgcg 4320 cggcggtcac tagaggctca tctccgccga acttcatgac cagcatgaag ggcacgagct 4380 gcttcccaaa ggcccccatc caagtatagg tctctacatc gtaggtgaca aagagacgct 4440 cggtgcgagg atgcgagccg atcgggaaga actggatctc ccgccaccag ttggaggagt 4500 ggctgttgat gtggtgaaag tagaagtccc tgcgacgggc cgaacactcg tgctggcttt 4560 tgtaaaaacg tgcgcagtac tggcagcggt gcacgggctg tacatcctgc acgaggttga 4620 cctgacgacc gcgcacaagg aagcagagtg ggaatttgag cccctcgcct ggcgggtttg 4680 gctggtggtc ttctacttcg gctgcttgtc cttgaccgtc tggctgctcg aggggagtta 4740 cggtggatcg gaccaccacg ccgcgcgagc ccaaagtcca gatgtccgcg cgcggcggtc 4800 ggagcttgat gacaacatcg cgcagatggg agctgtccat ggtctggagc tcccgcggcg 4860 tcaggtcagg cgggagctcc tgcaggttta cctcgcatag ccgggtcagg gcgcgggcta 4920 ggtccaggtg atacctgatt tccaggggct ggttggtggc ggcgtcgatg gcttgcaaga 4980 ggccgcatcc ccgcggcgcg actacggtac cgcgcggcgg gcggtgggcc gcgggggtgt 5040 ccttggatga tgcatctaaa agcggtgacg cgggcgggcc cccggaggta gggggggctc 5100 gggacccgcc gggagagggg gcaggggcac gtcggcgccg cgcgcgggca ggagctggtg 5160 ctgcgcgcgg aggttgctgg cgaacgcgac gacgcggcgg ttgatctcct gaatctggcg 5220 cctctgcgtg aagacgacgg gcccggtgag cttgaacctg aaagagagtt cgacagaatc 5280 aatttcggtg tcgttgacgg cggcctggcg caaaatctcc tgcacgtctc ctgagttgtc 5340 ttgataggcg atctcggcca tgaactgctc gatctcttcc tcctggagat ctccgcgtcc 5400 ggctcgctcc acggtggcgg cgaggtcgtt ggagatgcgg gccatgagct gcgagaaggc 5460 gttgaggcct ccctcgttcc agacgcggct gtagaccacg cccccttcgg catcgcgggc 5520 gcgcatgacc acctgcgcga gattgagctc cacgtgccgg gcgaagacgg cgtagtttcg 5580 caggcgctga aagaggtagt tgagggtggt ggcggtgtgt tctgccacga agaagtacat 5640 aacccagcgc cgcaacgtgg attcgttgat atcccccaag gcctcaaggc gctccatggc 5700 ctcgtagaag tccacggcga agttgaaaaa ctgggagttg cgcgccgaca cggttaactc 5760 ctcctccaga agacggatga gctcggcgac agtgtcgcgc acctcgcgct caaaggctac 5820 aggggcctct tcttcttctt caatctcctc ttccataagg gcctcccctt cttcttcttc 5880 tggcggcggt gggggagggg ggacacggcg gcgacgacgg cgcaccggga ggcggtcgac 5940 aaagcgctcg atcatctccc cgcggcgacg gcgcatggtc tcggtgacgg cgcggccgtt 6000 ctcgcggggg cgcagttgga agacgccgcc cgtcatgtcc cggttatggg ttggcggggg 6060 gctgccgtgc ggcagggata cggcgctaac gatgcatctc aacaattgtt gtgtaggtac 6120 tccgccaccg agggacctga gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa 6180 ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc gtggcgggcg gcagcgggcg 6240 gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg taattaaagt aggcggtctt 6300 gagacggcgg atggtcgaca gaagcaccat gtccttgggt ccggcctgct gaatgcgcag 6360 gcggtcggcc atgccccagg cttcgttttg acatcggcgc aggtctttgt agtagtcttg 6420 catgagcctt tctaccggca cttcttcttc tccttcctct tgtcctgcat ctcttgcatc 6480 tatcgctgcg gcggcggcgg agtttggccg taggtggcgc cctcttcctc ccatgcgtgt 6540 gccccgaag cccctcatcg gctgaagcag ggccaggtcg gcgacaacgc gctcggctaa 6600 tatggcctgc tgcacctgcg tgagggtaga ctggaagtcg tccatgtcca caaagcggtg 6660 gtatgcgccc gtgttgatgg tgtaagtgca gttggccata acggaccagt taacggtctg 6720 gtgacccggc tgcgagagct cggtgtacct gagacgcgag taagcccttg agtcaaagac 6780 gtagtcgttg caagtccgca ccaggtactg gtatcccacc aaaaagtgcg gcggcggctg 6840 gcggtagagg ggccagcgta gggtggccgg ggctccgggg gcgaggtctt ccaacataag 6900 gcgatgatat ccgtagatgt acctggacat ccaggtgatg ccggcggcgg tggtggaggc 6960 gcgcggaaag tcacggacgc ggttccagat gttgcgcagc ggcaaaaagt gctccatggt 7020 cgggacgctc tggccggtca ggcgcgcgca gtcgttgacg ctctagaccg tgcaaaagga 7080 gagcctgtaa gcgggcactc ttccgtggtc tggtggataa attcgcaagg gtatcatggc 7140 ggacgaccgg ggttcgaacc ccggatccgg ccgtccgccg tgatccatgc ggttaccgcc 7200 cgcgtgtcga acccaggtgt gcgacgtcag acaacggggg agcgctcctt ttggcttcct 7260 tccaggcgcg gcggatgctg cgctagcttt tttggccact ggccgcgcgc ggcgtaagcg 7320 gttaggctgg aaagcgaaag cattaagtgg ctcgctccct gtagccggag ggttattttc 7380 caagggttga gtcgcgggac ccccggttcg agtctcgggc cggccggact gcggcgaacg 7440 ggggtttgcc tccccgtcat gcaagacccc gcttgcaaat tcctccggaa acagggacga 7500 gccccttttt tgcttttccc agatgcatcc ggtgctgcgg cagatgcgcc cccctcctca 7560 gcagcggcaa gagcaagagc agcggcagac atgcagggca ccctcccctt ctcctaccgc 7620 gtcaggaggg gcaacatccg cggctgacgc ggcggcagat ggtgattacg aacccccgcg 7680 gcgccggacc cggcactact tggacttgga ggagggcgag ggcctggcgc ggctaggagc 7740 gccctctcct gagcgacacc caagggtgca gctgaagcgt gacacgcgcg aggcgtacgt 7800 gccgcggcag aacctgtttc gcgaccgcga gggagaggag cccgaggaga tgcgggatcg 7860 aaagttccat gcagggcgcg agttgcggca tggcctgaac cgcgagcggt tgctgcgcga 7920 ggaggacttt gagcccgacg cgcggaccgg gattagtccc gcgcgcgcac acgtggcggc 7980 cgccgacctg gtaaccgcgt acgagcagac ggtgaaccag gagattaact ttcaaaaaag 8040 ctttaacaac cacgtgcgca cgcttgtggc gcgcgaggag gtggctatag gactgatgca 8100 tctgtgggac tttgtaagcg cgctggagca aaacccaaat agcaagccgc tcatggcgca 8160 gctgttcctt atagtgcagc acagcaggga caacgaggca ttcagggatg cgctgctaaa 8220 catagtagag cccgagggcc gctggctgct cgatttgata aacattctgc agagcatagt 8280 ggtgcaggag cgcagcttga gcctggctga caaggtggcc gccattaact attccatgct 8340 cagtctgggc aagttttacg cccgcaagat ataccatacc ccttacgttc ccatagacaa 8400 ggaggtaaag atcgaggggt tctacatgcg catggcgctg aaggtgctta ccttgagcga 8460 cgacctgggc gtttatcgca acgagcgcat ccacaaggcc gtgagcgtga gccggcggcg 8520 cgagctcagc gaccgcgagc tgatgcacag cctgcaaagg gccctggctg gcacgggcag 8580 cggcgataga gaggccgagt cctactttga cgcgggcgct gacctgcgct gggccccaag 8640 ccgacgcgcc ctggaggcag ctggggccgg acctgggctg gcggtggcac ccgcgcgcgc 8700 tggcaacgtc ggcggcgtgg aggaatatga cgaggacgat gagtacgagc cagaggacgg 8760 cgagtactaa gcggtgatgt ttctgatcag atgatgcaag acgcaacgga cccggcggtg 8820 cgggcggcgc tgcagagcca gccgtccggc cttaactcca cggacgactg gcgccaggtc 8880 atggaccgca tcatgtcgct gactgcgcgc aaccctgacg cgttccggca gcagccgcag 8940 gccaaccggc tctccgcaat tctggaagcg gtggtcccgg cgcgcgcaaa ccccacgcac 9000 gagaaggtgc tggcgatcgt aaacccgctg gccgaaaaca gggccatccg gcccgatgag 9060 gccggcctgg tctacgacgc gctgcttcag cgcgtggctc gttacaacag cagcaacgtg 9120 cagaccaacc tggaccggct ggtgggggat gtgcgcgagg ccgtggcgca gcgtgagcgc 9180 gcgcagcagc agggcaacct gggctccatg gttgcactaa acgccttcct gagtacacag 9240 cccgccaacg tgccgcgggg acaggaggac tacaccaact ttgtgagcgc actgcggcta 9300 atggtgactg agacaccgca aagtgaggtg tatcagtccg ggccagacta ttttttccag 9360 accagtagac aaggcctgca gaccgtaaac ctgagccagg ctttcaagaa cttgcagggg 9420 ctgtgggggg tgcgggctcc cacaggcgac cgcgcgaccg tgtctagctt gctgacgccc 9480 aactcgcgcc tgttgctgct gctaatagcg cccttcacgg acagtggcag cgtgtcccgg 9540 gacacatacc taggtcactt gctgacactg taccgcgagg ccataggtca ggcgcatgtg 9600 gacgagcata ctttccagga gattacaagt gttagccgcg cgctggggca ggaggacacg 9660 ggcagcctgg aggcaaccct gaactacctg ctgaccaacc ggcggcaaaa aatcccctcg 9720 ttgcacagtt taaacagcga ggaggagcgc attttgcgct atgtgcagca gagcgtgagc 9780 cttaacctga tgcgcgacgg ggtaacgccc agcgtggcgc tggacatgac cgcgcgcaac 9840 atggaaccgg gcatgtatgc ctcaaaccgg ccgtttatca atcgcctaat ggactacttg 9900 catcgcgcgg ccgccgtgaa ccccgagtat ttcaccaatg ccatcttgaa cccgcactgg 9960 ctaccgcccc ctggtttcta caccggggga ttcgaggtgc ccgagggtaa cgatggattc 10020 ctctgggacg acatagacga cagcgtgttt tccccgcaac cgcagaccct gctagagttg 10080 caacaacgcg agcaggcaga ggcggcgctg cgaaaggaaa gcttccgcag gccaagcagc 10140 ttgtccgatc taggcgctgc ggccccgcgg tcagatgcta gtagcccatt tccaagcttg 10200 atagggtctc ttaccagcac tcgcaccacc cgcccgcgcc tgctgggcga ggaggagtac 10260 ctaaacaact cgctgctgca gccgcagcgc gaaaagaacc tgcctccggc gtttcccaac 10320 aacgggatag agagcctagt ggacaagatg agtagatgga agacgtatgc gcaggagcac 10380 agggatgtgc ccggcccgcg cccgcccacc cgtcgtcaaa ggcacgaccg tcagcggggt 10440 ctggtgtggg aggacgatga ctcggcagac gacagcagcg tcttggattt gggagggag 10500 ggcaacccgt ttgcacacct tcgccccagg ctggggagaa tgttttaaaa aaagcatgat 10560 gcaaaataaa aaactcacca aggcnatggc accgagcgtt ggttttcttg tattcccctt 10620 agtatgcggc gcgcggcgat gtatgaggaa ggtcctcctc cctcctacga gagcgtggtg 10680 agcgcggcgc cagtggcggc ggcgctgggt tcacccttcg atgctcccct ggacccgccg 10740 ttcgtgcctc cgcggtacct gcggcctacc ggggggagaa acagcatccg ttactctgag 10800 ttggcacccc tattcgacac cacccgtgtg taccttgtgg acaacaagtc aacggatgtg 10860 gcatccctga actaccagaa cgaccacagc aactttctaa ccacggtcat tcaaaacaat 10920 gactacagcc cgggggaggc aagcacacag accatcaatc ttgacgaccg gtcgcactgg 10980 ggcggcgacc tgaaaaccat cctgcatacc aacatgccaa atgtgaacga gttcatgttt 11040 accaataagt ttaaggcgcg ggtgatggtg tcgcgctcgc ttactaagga caaacaggtg 11100 ggctgaaat acgagtgggt ggagttcacg ctgcccgagg gcaactactc cgagaccatg 11160 accatagacc ttatgaacaa cgcgatcgtg gagcactact tgaaagtggg caggcagaac 11220 ggggttctgg aaagcgacat cggggtaaag tttgacaccc gcaacttcag actggggttt 11280 gacccagtca ctggtcttgt catgcctggg gtatataca acgaagcctt ccatccagac 11340 atcattttgc tgccaggatg cggggtggac ttcacccaca gccgcctgag caacttgttg 11400 ggcatccgca agcggcaacc cttccaggag ggctttagga tcacctacga tgacctggag 11460 ggtggtaaca ttcccgcact gttggatgtg gacgcctacc aggcaagctt gaaagatgac 11520 accgaacagg gcgggggtgg cgcaggcggc ggcaacaaca gtggcagcgg cgcggaagag 11580 aactccaacg cggcagctgc ggcaatgcag ccggtggagg acatgaacga tcatgccatt 11640 cgcggcgaca cctttgccac acgggcggag gagaagcgcg ctgaggccga ggcagcggcc 11700 gaagctgccg cccccgctgc ggaggctgca caacccgagg tcgagaagcc tcagaagaaa 11760 ccggtgatta aacccctgac agaggacagc aagaaacgca gttacaacct aataagcaat 11820 gacagcacct tcacccagta ccgcagctgg taccttgcat acaactacgg cgaccctcag 11880 gccgggatcc gctcatggac cctgctttgc actcctgacg taacctgcgg ctcggagcag 11940 gtatactggt cgttgcccga catgatgcaa gaccccgtga ccttccgctc cacgcgccag 12000 atcagcaact ttccggtggt gggcgccgag ctgttgcccg tgcactccaa gagcttctac 12060 aacgaccagg ccgtctactc ccagctcatc cgccagttta cctctctgac ccacgtgttc 12120 aatcgctttc ccgagaacca gattttggcg cgcccgccag cccccaccat caccaccgtc 12180 agtgaaaacg ttcctgctct cacagatcac gggacgctac cgctgcgcaa cagcatcgga 12240 ggagtccagc gagtgaccat tactgacgcc agacgccgca cctgccccta cgtttacaag 12300 gccctgggca tagtctcgcc gcgcgtccta tcgagccgca ctttttgagc aagcatgtcc 12360 atccttatat cgcccagcaa taacacaggc tggggcctgc gcttcccaag caagatgttt 12420 ggcggggcca agaagcgctc cgaccaacac ccagtgcgcg tgcgcgggca ctaccgcgcg 12480 ccctggggcg cgcacaaacg cggccgcact gggcgcacca ccgtcgatga cgccatcgac 12540 gcggtggtgg aggaggcgcg caactacacg cccacgccgc cgccagtgtc caccgtggac 12600 gcggccattc agaccgtggt gcgcggagcc cggcgctacg ctaaaatgaa gagacggcgg 12660 aggcgcgtag cacgtcgcca ccgccgccga cccggcactg ccgcccaacg cgcggcggcg 12720 gccctgctta accgcgcacg tcgcaccggc cgacgggcgg ccatgcgagc cgctcgaagg 12780 ctggccgcgg gtattgtcac tgtgcccccc aggtccaggc gacgagcggc cgccgcagca 12840 gccgcggcca ttagtgttat gactcagggt cgcaggggca acgtgtactg ggtgcgcgac 12900 tcggttagcg gcctgcgcgt gcccgtgcgc acccgccccc cgcgcaacta gattgcaata 12960 aaaaactact tagactcgta ctgttgtatg tatccagcgg cggcggcgcg catcgaagct 13020 atgtccaagc gcaaaatcaa agaagagatg ctccaggtca tcgcgccgga gatctatggc 13080 cccccgaaga aggaagagca ggattacaag ccccgaaagc taaagcgggt caaaaagaaa 13140 aagaaagatg atgatgatga tgaacttgac gacgaggtgg aactgttgca cgcgaccgcg 13200 cccaggcgac gggtacagtg gaaaggtcga cgcgtaagac gtgttttgcg acccggcacc 13260 accgtagtct ttacgcccgg tgagcgctcc acccgcacct acaagcgcgt gtatgatgag 13320 gtgtacggcg acgaggacct gcttgagcag gccaacgagc gcctcgggga gtttgcctac 13380 ggaaagcggc ataaggacat gctggcgttg ccgctggacg agggcaaccc aacacctagc 13440 ctaaagcccg tgacactgca gcaggtgctg cccgcgcttg caccgtccga agaaaagcgc 13500 ggcctaaagc gcgagtctgg tgacttggca cccaccgtgc agctgatggt acccaagcgt 13560 cagcgactgg aagatgtctt ggaaaaaatg accgtggagc ctgggctgga gcccgaggtc 13620 cgcgtgcggc caatcaagca ggtggcaccg ggactgggcg tgcagaccgt ggacgttcag 13680 atacccacca ccagtagcac tagtattgcc actgccacag agggcatgga gacacaaacg 13740 tccccggttg cctcggcggt ggcagatgcc gcggtgcagg cggccgctgc ggccgcgtcc 13800 aagacctcta cggaggtgca aacggacccg tggatgtttc gtgtttcagc cccccggcgt 13860 ccgcgccgtt caaggaagta cggcgccgcc agcgcgctac tgcccgaata tgccctacat 13920 ccttccatcg cgcctacccc cggctatcgt ggctacacct accgccccag aagacgagca 13980 actacccgac gccgaaccac cactggaacc cgccgccgcc gtcgccgtcg ccagcccgtg 14040 ctggccccga tttccgtgcg cagggtggct cgcgaaggag gcaggaccct ggtgctgcca 14100 acagcgcgct accaccccag catcgtttaa aagccggtct ttgtggttct tgcagatatg 14160 gccctcacct gccgcctccg tttcccggtg ccgggattcc gaggaagaat gcaccgtagg 14220 aggggcatgg ccggccacgg cctgacgggc ggcatgcgtc gtgcgcacca ccggcggcgg 14280 cgcgcgtcgc accgtcgcat gcgcgccggt atcctgcccc tccttattcc actgatcgcc 14340 gcggcgattg gcgccgtgcc cggaattgca tccgtggcct tgcaggcgca gagacactga 14400 ttaaaaacaa gttacatgtg gaaaaatcaa aataaaagtc tggactctca cgctcgcttg 14460 gtcctgtaac tattttgtag aatggaagac atcaactttg cgtcactggc cccgcgacac 14520 ggctcgcgcc cgttcatggg aaactggcaa gatatcggca ccagcaatat gagcggtggc 14580 gccttcagct ggggctcgct gtggagcggc attaaaaatt tcggttccgc cgttaagaac 14640 tatggcagca aagcctggaa cagcagcaca ggccagatgc tgagggacaa gttgaaagag 14700 caaaatttcc aacaaaaggt ggtagatggc ctggcctctg gcattagcgg ggtggtggac 14760 ctggccaacc aggcagtgca aaataagatt aacagtaagc ttgatccccg ccctcccgta 14820 gaggagcctc caccggccgt ggagacagtg tctccagagg ggcgtggcga aaagcgtccg 14880 cgacccgaca gggaagaaac tctggtgacg caaatagacg agcctccctc gtacgaggag 14940 gcactaaagc aaggcctgcc caccacccgt cccatcgcgc ccatggctac cggagtgctg 15000 ggccagcaca cacccgtaac gctggacctg cctccccccg ccgacaccca gcagaaacct 15060 gtgctgccag gcccgtccgc cgttgttgta acccgtccta gccgcgggtc cctgcgccgc 15120 gccgccagcg gtccgcgatc gttgcggccc gtagccagtg gcaactggca aagcacactg 15180 aacagcatcg tgggtttggg ggtgcaatcc ctgaagcgcc gacgatgctt ctgatagcta 15240 acgtgtcgta tgtgtgtcat gtatgcgtcc atgtcgccgc cagaggagct gctgagccgc 15300 cgcgcgcccg ctttccaaga tggctacccc ttcgatgatg ccgcagtggt cttacatgca 15360 catctcgggc caggacgcct cggagtacct gagccccggg ctggtgcagt tcgcccgcgc 15420 caccgagacg tacttcagcc tgaataacaa gtttagaaac cccacggtgg cgcctacgca 15480 cgacgtgacc acagaccggt ctcagcgttt gacgctgcgg ttcatccccg tggaccgcga 15540 ggatactgcg tactcgtaca aggcgcggtt caccctagct gtgggtgata accgtgtgct 15600 agacatggct tccacgtact ttgacatccg cggcgtgctg gacaggggcc ctacttttaa 15660 gccctactct ggcactgcct acaacgcact ggcccccaag ggtgccccca actcgtgcga 15720 gtgggaacaa aatgaaactg cacaagtgga tgctcaagaa cttgacgaag aggagaatga 15780 agccaatgaa gctcaggcgc gagaacagga acaagctaag aaaacccatg tatatgccca 15840 ggctccactg tccggaataa aaataactaa agaaggtcta caaataggaa ctgccgacgc 15900 cacagtagca ggtgccggca aagaaatttt cgcagacaaa acttttcaac ctgaaccaca 15960 agtaggagaa tctcaatgga acgaagcgga tgccacagca gctggtggaa gggttcttaa 16020 aaagacaact cccatgaaac cctgctatgg ctcatacgct agacccacca attccaacgg 16080 cggacagggc gttatggttg aacaaaatgg taaattggaa agtcaagtcg aaatgcaatt 16140 tttttccaca tccacaaatg ccacaaatga agttaacaat atacaaccaa cagttgtatt 16200 gtacagcgaa gatgtaaaca tggaaactcc agatactcat ctttcttata aacctaaaat 16260 gggggataaa aatgccaaag tcatgcttgg acaacaagca atgccaaaca gaccaaatta 16320 cattgctttt agagacaatt ttattggtct catgtattac aacagcacag gtaacatggg 16380 tgtccttgct ggtcaggcat cgcagttgaa cgctgttgta gatttgcaag acagaaacac 16440 agagctgtcc taccagcttt tgcttgattc aattggcgac agaacaagat acttttcaat 16500 gtggaatcaa gctgttgaca gctatgatcc agatgtcaga attattgaga accatggaac 16560 tgaggatgag ttgccaaatt attgctttcc tcttggtgga attgggatta ctgacacttt 16620 tcaagctgtt aaaacaactg ctgctaacgg ggaccaaggc aatactacct ggcaaaaaga 16680 ttcaacattt gcagaacgca atgaaatagg ggtgggaaat aactttgcca tggaaattaa 16740 cctgaatgcc aacctatgga gaaatttcct ttactccaat attgcgctgt acctgccaga 16800 caagctaaaa tacaacccca ccaatgtgga aatatctgac aaccccaaca cctacgacta 16860 catgaacaag cgagtggtgg ctcctgggct tgtagactgc tacattaacc ttggggcgcg 16920 ctggtctctg gactacatgg acaacgttaa tccctttaac cacccccgcc atgcgggcct 16980 gcgttaccgc tccatgttgt tgggaaacgg ccgctacgtg ccctttcaca ttcaggtgcc 17040 ccaaaagttt tttgccatta aaaacctcct cctcctgcca ggctcataca catatgaatg 17100 gaacttcagg aaggatgtta acatggttct gcagagctct ctgggaaacg accttagagt 17160 tgacggggct agcattaagt ttgacagcat ttgtctttac gccaccttct tccccatggc 17220 ccacaacacg gcctccacgc tggaagccat gctcagaaat gacaccaacg accagtcctt 17280 taatgactac ctttccgccg ccaacatgct atatcccata cccgccaacg ccaccaacgt 17340 gcccatctcc atcccatcgc gcaactgggc agcatttcgc ggttgggcct tcacacgctt 17400 gaagacaaag gaaacccctt ccctgggatc aggctacgac ccttactaca cctactctgg 17460 ctccatacca taccttgacg gaaccttcta tcttaatcac acctttaaga aggtggccat 17520 tacttttgg tcttctgtta gctggccggg caacgaccgc ctgcttactc ccaatgagtt 17580 tgagattaag cgctcagttg acggggaggg ctataacgta gctcagtgca acatgacaaa 17640 ggactggttc ctagtgcaga tgttggccaa ctacaatatt ggctaccagg gcttctacat 17700 tccagaaagc tacaaagacc gcatgtactc gttcttcaga aacttccagc ccatgagccg 17760 gcaagtggtg gacgatacta aatacaaaga ttatcagcag gttggaatta tccaccagca 17820 taacaactca ggcttcgtag gctacctcgc tcccaccatg cgcgagggac aagcttaccc 17880 cgctaatgtt ccctacccac taataggcaa aaccgcggtt gatagtatta cccagaaaaa 17940 gtttctttgc gaccgcaccc tgtggcgcat ccccttctcc agtaacttta tgtccatggg 18000 tgcgctcaca gacctgggcc aaaaccttct ctacgcaaac tccgcccacg cgctagacat 18060 gacctttgag gtggatccca tggacgagcc cacccttctt tatgttttgt ttgaagtctt 18120 tgacgtggtc cgtgtgcacc agccgcaccg cggcgtcatc gagaccgtgt acctgcgcac 18180 gcccttctcg gccggcaacg ccacaacata aagaagcaag caacatcaac aacagctgcc 18240 gccatgggct ccagtgagca ggaactgaaa gccattgtca aagatcttgg ttgtgggcca 18300 tattttttgg gcacctatga caagcgcttc ccaggctttg tttccccaca caagctcgcc 18360 tgcgccatag ttaacacggc cggtcgcgag actgggggcg tacactggat ggcctttgcc 18420 tggaacccgc gctcaaaaac atgctacctc tttgagccct ttggcttttc tgaccaacgt 18480 ctcaagcagg tttaccagtt tgagtacgag tcactcctgc gccgtagcgc cattgcctct 18540 tcccccgacc gctgtataac gctggaaaag tccacccaaa gcgtgcaggg gcccaactcg 18600 gccgcctgtg gcctattctg ctgcatgttt ctccacgcct ttgccaactg gccccaaact 18660 cccatggatc acaaccccac catgaacctt attaccgggg tacccaactc catgcttaac 18720 agtccccagg tacagcccac cctgcgccgc aaccaggaac agctctacag cttcctggag 18780 cgccactcgc cctacttccg cagccacagt gcgcaaatta ggagcgccac ttctttttgt 18840 cacttgaaaa acatgtaaaa ataatgtact aggagacact ttcaataaag gcaaatgttt 18900 ttatttgtac actctcgggt gattatttac ccccaccctt gccgtctgcg ccgtttaaaa 18960 atcaaagggg ttctgccgcg catcgctatg cgccactggc agggacacgt tgcgatactg 19020 gtgtttagtg ctccacttaa actcaggcac aaccatccgc ggcagctcgg tgaagttttc 19080 actccacagg ctgcgcacca tcaccaacgc gtttagcagg tcgggcgccg atatcttgaa 19140 gtcgcagttg gggcctccgc cctgcgcgcg cgagttgcga tacacagggt tacagcactg 19200 gaacactatc agcgccgggt ggtgcacgct ggccagcacg ctcttgtcgg agatcanatc 19260 cgcgtccagg tcctccgcgt tgctcagggc gaacggagtc aactttggta gctgccttcc 19320 caaaaagggt gcatgcccag gctttgagtt gcactcgcac cgtagtggca tcagaaggtg 19380 accgtgccca gtctgggcgt taggatacag cgcctgcatg aaagccttga tctgcttaaa 19440 agccacctga gcctttgcgc cttcagagaa gaacatgccg caagacttgc cggaaaactg 19500 attggccgga caggccgcgt catgcacgca gcaccttgcg tcggtgttgg agatctgcac 19560 cacatttcgg ccccaccggt tcttcacgat cttggccttg ctagactgct ccttcagcgc 19620 gcgctgcccg ttttcgctcg tcacatccat ttcaatcacg tgctccttat ttatcataat 19680 gctcccgtgt agacacttaa gctcgccttc gatctcagcg cagcggtgca gccacaacgc 19740 gcagcccgtg ggctcgtggt gcttgtaggt tacctctgca aacgactgca ggtacgcctg 19800 caggaatcgc cccatcatcg tcacaaaggt cttgttgctg gtgaaggtca gctgcaaccc 19860 gcggtgctcc tcgtttagcc aggtcttgca tacggccgcc agagcttcca cttggtcagg 19920 cagtagcttg aagtttgcct ttagatcgtt atccacgtgg tacttgtcca tcaacgcgcg 19980 cgcagcctcc atgcccttct cccacgcaga cacgatcggc aggctcagcg ggtttatcac 20040 cgtgctttca ctttccgctt cactggactc ttccttttcc tcttgcatcc gcataccccg 20100 cgccactggg tcgtcttcat tcagccgccg caccgtgcgc ttacctccct tgccgtgctt 20160 gattagcacc ggtgggttgc tgaaacccac catttgtagc gccacatctt ctctttcttc 20220 ctcgctgtcc acgatcacct ctggggatgg cgggcgctcg ggcttgggag aggggcgctt 20280 ctttttcttt ttggacgcaa tggccaaatc cgccgtcgag gtcgatggcc gcgggctggg 20340 tgtgcgcggc accagcgcat cttgtgacga gtcttcttcg tcctcggact cgagacgccg 20400 cctcagccgc ttttttgggg gcgcgcgggg aggcggcggc gacggcgacg gggacgagac 20460 gtcctccatg gttggtggac gtcgcgccgc accgcgtccg cgctcggggg tggtttcgcg 20520 ctgctcctct tcccgactgg ccatttcctt ctcctatagg cagaaaaaga tcatggagtc 20580 agtcgagaag gaggacagcc taaccgcccc ctttgagttc gccaccaccg cctccaccga 20640 tgccgccaac gcgcctacca ccttccccgt cgaggcaccc ccgcttgagg aggaggaagt 20700 gattatcgag caggacccag gttttgtaag cgaagacgac gaagatcgct cagtaccaac 20760 agaggataaa aagcaagacc aggacgacgc agaggcaaac gaggaacaag tcgggcgggg 20820 ggaccaaagg catggcgact acctagatgt gggagacgac gtgctgttga agcatctgca 20880 gcgccagtgc gccattatct gcgacgcgtt gcaagagcgc agcgatgtgc ccctcgccat 20940 agcggatgtc agccttgcct acgaacgcca cctgttctca ccgcgcgtac cccccaaacg 21000 ccaagaaaac ggcacatgcg agcccaaccc gcgcctcaac ttctaccccg tatttgccgt 21060 gccagaggtg cttgccacct atcacatctt tttccaaaac tgcaagatac ccctatcctg 21120 ccgtgccaac cgcagccgag cggacaagca gctggccttg cggcagggcg ctgtcatacc 21180 tgatatcgcc tcgctcgacg aagtgccaaa aatctttgag ggtcttggac gcgacgagaa 21240 gcgcgcggca aacgctctgc aacaagaaaa cagcgaaaat gaaagtcact gtggagtgct 21300 ggtggaactt gagggtgaca acgcgcgcct agccgtgctg aaacgcagca tcgaggtcac 21360 ccactttgcc tacccggcac ttaacctacc ccccaaggtt atgagcacag tcatgagcga 21420 gctgatcgtg cgccgtgcac gacccctgga gagggatgca aacttgcaag aacaaaccga 21480 ggagggccta cccgcagttg gcgatgagca gctggcgcgc tggcttgaga cgcgcgagcc 21540 tgccgacttg gaggagcgac gcaagctaat gatggccgca gtgcttgtta ccgtggagct 21600 tgagtgcatg cagcggttct ttgctgaccc ggagatgcag cgcaagctag aggaaacgtt 21660 gcactacacc tttcgccagg gctacgtgcg ccaggcctgc aaaatttcca acgtggagct 21720 ctgcaacctg gtctcctacc ttggaatttt gcacgaaaac cgccttgggc aaaacgtgct 21780 tcattccacg ctcaagggcg aggcgcgccg cgactacgtc cgcgactgcg tttacttatt 21840 tctgtgctac acctggcaaa cggccatggg cgtgtggcag cagtgcctgg aggagcgcaa 21900 cctgaaggag ctgcagaagc tgctaaagca aaacttgaag gacctatgga cggccttcaa 21960 cgagcgctcc gtggccgcgc acctggcgga cattatcttc cccgaacgcc tgcttaaaac 22020 cctgcaacag ggtctgccag acttcaccag tcaaagcatg ttgcaaaact ttaggaactt 22080 tatcctagag cgttcaggaa ttctgcccgc cacctgctgt gcgcttccta gcgactttgt 22140 gcccattaag tccgtgaat gccctccgcc gctttggggt cactgctacc ttntgcagct 22200 agccaactac cttgcctacc actccgacat catggaagac gtgagcggtg acggcctact 22260 ggagtgtcac tgtcgctgca acctatgcac cccgcaccgc tccctggtct gcaattcaca 22320 actgcttagc gaaagtcaaa ttatcggtac ctttgagctg cagggtccct cgcctgacga 22380 aaagtccgcg gctccggggt tgaaactcac tccggggctg tggacgtcgg cttaccttcg 22440 caaatttgta cctgaggact accacgccca cgagattagg ttctacgaag accaatcccg 22500 cccgccaaat gcggagctta ccgcctgcgt cattacccag ggccacatcc ttggccaatt 22560 gcaagccatt aacaaagccc gccaagagtt tctgctacga aagggacggg gggtttactt 22620 ggacccccag tccggcgagg agctcaaccc aatccccccg ccgccgcagc cctatcagca 22680 gccgcgggcc cttgcttccc aggatggcac ccaaaaagaa gctgcagctg ccgccgccgc 22740 cacccacgga cgaggaggaa tactgggaca gtcaggcaga ggaggttttg gacgaggagg 22800 aggagatgat ggaagactgg gacagcctag acgaggaagc ttccgaggcc gaagaggtgt 22860 cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc ggcgccccag aaatcggcaa 22920 ccgttcccag cattgctaca acctccgctc ctcaggcgcc gccggcactg cccgttcgcc 22980 gacccaaccg tagatgggac accactggaa ccagggccgg taagtctaag cagccgccgc 23040 cgttagccca agagcaacaa cagcgccaag gctaccgctc gtggcgcgtg cacaagaacg 23100 ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc cttcgcccgc cgctttcttc 23160 tctaccatca cggcgtggcc ttcccccgta acatcctgca ttactaccgt catctctaca 23220 gcccctactg caccggcggc agcggcagca acagcagcgg ccacgcagaa gcaaaggcga 23280 ccggatagca agactctgac aaagcccaag aaatccacag cggcggcagc agcaggagga 23340 ggagcactgc gtctggcgcc caacgaaccc gtatcgaccc gcgagcttag aaacaggatt 23400 tttcccactc tgtatgctat atttcaacag agcaggggcc aagaacaaga gctgaaaata 23460 aaaaacaggt ctctgcgctc cctcacccgc agctgcctgt atcacaaaag cgaagatcag 23520 cttcggcgca cgctggaaga cgcggaggct ctcttcagca aatactgcgc gctgactctt 23580 aaggactagt ttcgcgccct ttctcaaatt taagcgcgaa aactacgtca tctccagcgg 23640 ccacacccgg cgccagcacc tgtcgtcagc gccattatga gcaaggaaat tcccacgccc 23700 tacatgtgga gttaccagcc acaaatggga cttgcggctg gagctgccca agactactca 23760 acccgaataa actacatgag cgcgggaccc cacatgatat cccgggtcaa cggaatccgc 23820 gcccaccgaa accgaattct cctcgaacag gcggctatta ccaccacacc tcgtaataac 23880 cttaatcccc gtagttggcc cgctgccctg gtgtaccagg aaagtcccgc tcccaccact 23940 gtggtacttc ccagagacgc ccaggccgaa gttcagatga ctaactcagg ggcgcagctt 24000 gcgggcggct ttcgtcacag ggtgcggtcg cccgggcagg gtataactca cctgaaaatc 24060 agagggcgag gtattcagct caacgacgag tcggtgagct cctctcttgg tctccgtccg 24120 gacgggacat ttcagatcgg cggcgcgtgg ccgctcttca tttacgcccc gtcaggcgat 24180 cctaactctg cagacctcgt cctcggagcc gcgctccgga ggcattggaa ctctacaatt 24240 tattgaggag ttcgtgcctt cggtttactt caaccccttt tctggacctc ccggccacta 24300 cccggaccag tttattccca actttgacgc ggtaaaagac tcggcggacg gctacgactg 24360 aatgaccagt ggagaggcag agcaactgcg cctgacacac ctcgaccact gccgccgcca 24420 caagtgcttt gcccgcggct ccggtgagtt ttgttacttt gaattgcccg aagagcatat 24480 cgagggcccg gcgcacggcg tccggctcac cacccaggta gagcttacac gtagcctgat 24540 tcgggagttt accaagcgcc ccctgctagt ggagcgggag cggggtccct gtgttctgac 24600 cgtggtttgc aactgtccta accctggatt acatcaagat ctttgttgtc atctctgtgc 24660 tgagtataat aaatacagaa attagaatct actggggctc ctgtcgccat cctgtgaacg 24720 ccaccgtttt tacccaccca aagcagacca aagcaaacct cacctccggt ttgcacaagc 24780 gt; tccagcgaga cgaagtaagt ttgccacaca accttctcgg cttcaactac accgtcaaga 24900 aaaacaccac caccaccctc ctcacctgcc gggaacgtac gagtgcgtca ccggttgctg 24960 cgcccacacc tacagcctga gcgtaaccag acattactcc cattttccca aaacaggagg 25020 tgagctcaac tcccggaact caggtcaaaa aagcattttg cggggtgctg ggatttttta 25080 attaagtata tgagcaattc aagtaactct acaagcttgt ctaatttttc tggaattggg 25140 gtcggggtta tccttactct tgtaattctg tttattctta tactagcact tctgtgcctt 25200 agggttgccg cctgctgcac gcacgtttgt acctattgtc agctttttaa acgctggggg 25260 cgacatccaa gatgaggtac atgattttag gcttgctcgc ccttgcggca gtctgcagcg 25320 ctgccaaaaa ggttgagttt aaggaaccag cttgcaatgt tacatttaaa tcagaagcta 25380 atgaatgcac tactcttata aaatgcacca cagaacatga aaagcttatt attcgccaca 25440 aagacaaaat tggcaagtat gctgtatatg ctatttggca gccaggtgac actaacgact 25500 ataatgtcac agtcttccaa ggtgaaaatc gtaaaacttt tatgtataaa tttccatttt 25560 atgaaatgtg cgatattacc atgtacatga gcaaacagta caagttgtgg cccccacaaa 25620 agtgtttaga gaacactggc accttttgtt ccaccgctct gcttattaca gcgcttgctt 25680 tggtatgtac cttactttat ctcaaataca aaagcagacg cagttttatt gatgaaaaga 25740 aaatgccttg attttccgct tgcttgtatt cccctggaca atttactcta tgtgggatat 25800 gcgccaggcg ggaaagatta tacccacaac cttcaaatca aactttcctg gacgttagcg 25860 cctgacttct gccagcgcct gcactgcaaa tttgatcaaa cccagcttca gcttgcctgc 25920 tccagagatg accggctcaa ccatcgcgcc cacaacggac tatcgcaaca ccactgctac 25980 cggactaaaa tctgccctaa atttacccca agttcatgcc tttgtcaatg actgggcgag 26040 cttgggcatg tggtggtttt ccatagcgct tatgtttgtt tgccttatta ttatgtggct 26100 tatttgttgc ctaaagcgca gacgcgccag accccccatc tataggccta tcattgtgct 26160 caacccacac aatgaaaaaa ttcatagatt ggacggtctc aaaccatgtt ctcttctttt 26220 acagtatgat taaatgagac atgattcctc gagtccttat attattgacc cttgttgcgc 26280 ttttctgtgc gtgctctaca ttggctgcgg tcgctcacat cgaagtagat tgcatcccac 26340 ctttcacagt ttacctgctt tacggatttg tcacccttat cctcatctgc agcctcgtca 26400 ctgtagtcat cgccttcatt cagttcattg actggatttg tgtgcgcatt gcgtacctta 26460 ggcaccatcc gcaatacaga gacaggacta tagctgatct tctcagaatt ctttaattat 26520 gaaacggatt gtcacttttg ttttgctgat tttctgcgcc ctacctgtgc tttgctccca 26580 aacctcagcg cctcccaaaa gacatatttc ctgcagattc actcaaatat ggaacattcc 26640 cagctgctac aacaaacaga gcgatttgtc agaagcctgg ttatacgcca tcatctctgt 26700 catggttttt tgcagtacca tttttgccct agccatatac ccataccttg acattggttg 26760 gaatgccata gatgccatga accaccctac tttcccagcg cccaatgtca taccactgca 26820 acaggttatt gccccaatca atcagcctcg ccccccttct cccaccccca ctgagattag 26880 ctactttaat ttgacaggtg gagatgactg aatctctaga tctagaattg gatggaatta 26940 acaccgaaca gcgcctacta gaaaggcgca aggcggcgtc cgagcgagaa cgcctaaaac 27000 aagaagttga agacatggtt aacctgcacc agtgtaaaag aggtatcttt tgtgtggtca 27060 agcaggccaa acttacctac gaaaaaacca ctaccggcaa ccgccttagc tacaagctac 27120 ccacccagcg ccaaaaactg gtgcttatgg tgggagaaaa acctatcacc gtcacccagc 27180 actcggcaga aacagaaggc tgcctgcact tcccctatca gggtccagag gacctctgca 27240 ctcttattaa aaccatgtgt ggcattagag atcttattcc attcaactaa caataaacac 27300 acaataaatt acttacttaa aatcagtcag caaatctttg tccagcttat tcagcatcac 27360 ctcctttccc tcctcccaac tctggtattt cagcagcctt ttagctgcga actttctcca 27420 aagtctaaat gggatgtcaa attcctcatg ttcttgtccc tccgcaccca ctatcttcat 27480 attgttgcag atgaaacgcg ccagaccgtc tgaagacacc ttcaaccctg tgtacccata 27540 tgacacggaa accggccctc caactgtgcc tttccttacc cctccctttg tgtcgccaaa 27600 tgggttccaa gaaagtcccc ccggagtgct ttctttgcgt ctttcagaac ctttggttac 27660 ctcacacggc atgcttgcgc taaaaatggg cagcggcctg tccctggatc aggcaggcaa 27720 ccttacatca aatacaatca ctgtttctca accgctaaaa aaaacaaagt ccaatataac 27780 tttggaaaca tccgcgcccc ttacagtcag ctcaggcgcc ctaaccatgg ccacaacttc 27840 gcctttggtg gtctctgaca acactcttac catgcaatca caagcaccgc taaccgtgca 27900 agactcaaaa cttagcattg ctaccaaaga gccacttaca gtgttagatg gaaaactggc 27960 cctgcagaca tcagcccccc tctctgccac tgataacaac gccctcacta tcactgcctc 28020 acctcctctt actactgcaa atggtagtct ggctgttacc atggaaaacc cactttacaa 28080 caacaatgga aaacttgggc tcaaaattgg cggtcctttg caagtggcca ccgactcaca 28140 tgcactaaca ctaggtactg gtcagggggt tgcagttcat aacaatttgc tacatacaaa 28200 agttacaggc gcaatagggt ttgatacatc tggcaacatg gaacttaaaa ctggagatgg 28260 cctctatgtg gatagcgccg gtcctaacca aaaactacat attaatctaa ataccacaaa 28320 aggccttgct tttgacaaca ccgcaataac aattaacgct ggaaaagggt tggaatttga 28380 aacagactcc tcaaacggaa atcccataaa aacaaaaatt ggatcaggca tacaatataa 28440 taccaatgga gctatggttg caaaacttgg aacaggcctc agttttgaca gctccggagc 28500 cataacaatg ggcagcataa acaatgacag acttactctt tggacaacac cagacccatc 28560 cccaaattgc agaattgctt cagataaaga ctgcaagcta actctggcgc taacaaaatg 28620 tggcagtcaa attttgggca ctgtttcagc tttggcagta tcaggtaata tggcctccat 28680 caatggaact ctaagcagtg taaacttggt tcttagattt gatgacaacg gagtgcttat 28740 gtcaaattca tcactggaca aacagtattg gaactttaga aacggggact ccactaacgg 28800 tcaaccatac acttatgctg ttgggtttat gccaaaccta aaagcttacc caaaaactca 28860 aagtaaaact gcaaaaagta atattgttag ccaggtgtat cttaatggtg acaagtctaa 28920 accattgcat tttactatta cgctaaatgg aacagatgaa accaaccaag taagcaaata 28980 ctcaatatca ttcagttggt cctggaacag tggacaatac actaatgaca aatttgccac 29040 caattcctat accttctcct acattgccca ggaataaaga atcgtgaacc tgttgcatgt 29100 tatgtttcaa cgtgtttatt tttcaattgc agaaaatttc aagtcatttt tcattcagta 29160 gtatagcccc accaccacat agcttatact aatcaccgta ccttaatcaa actcacagaa 29220 ccctagtatt caacctgcca cctccctccc aacacacaga gtacacagtc ctttctcccc 29280 ggctggcctt aaacagcatc atatcatggg taacagacat attcttaggt gttatattcc 29340 acacggtctc ctgtcgagcc aaacgctcat cagtgatgtt aataaactcc ccgggcagct 29400 cgcttaagtt catgtcgctg tccagctgct gagccacagg ctgctgtcca acttgcggtt 29460 gctcaacggg cggcgaagga gaagtccacg cctacatggg ggtagagtca taatcgtgca 29520 tcaggatagg gcggtggtgc tgcagcagcg cgcgaataaa ctgctgccgc cgccgctccg 29580 tcctgcagga atacaacatg gcagtggtct cctcagcgat gattcgcacc gcccgcagca 29640 taaggcgcct tgtcctccgg gcacagcagc gcaccctgat ctcacttaag tcagcacagt 29700 aactgcagca cagtaccaca atattgttta aaatcccaca gtgcaaggcg ctgtatccaa 29760 agctcatggc ggggaccaca gaacccacgt ggccatcata ccacaagcgc aggtagatta 29820 agtggcgacc cctcataaac acgctggaca taaacattac ctcttttggc atgttgtaat 29880 tcaccacctc ccggtaccat ataaacctct gattaaacat ggcgccatcc accaccatcc 29940 taaaccagct ggccaaaacc tgcccgccgg ctatgcactg cagggaaccg ggactggaac 30000 aatgacagtg gagagcccag gactcgtaac catggatcat catgctcgtc atgatatcaa 30060 tgttggcaca acacaggcac acgtgcatac acttcctcag gattacaagc tcctcccgcg 30120 tcagaaccat atcccaggga acaacccatt cctgaatcag cgtaaatccc acactgcagg 30180 gaagacctcg cacgtaactc acgttgtgca ttgtcaaagt gttacattcg ggcagcagcg 30240 gatgatcctc cagtatggta gcgcgtgtct ctgtctcaaa aggaggtagg cgatccctac 30300 tgtacggagt gcgccgagac aaccgagatc gtgttggtcg tagtgtcatg ccaaatggaa 30360 cgccggacgt agtcatattt cctgaagcaa aaccaggtgc gggcgtgaca aacagatctg 30420 cgtctccggt ctcgtcgctt agctcgctct gtgtagtagt tgtagtatat ccactctctc 30480 aaagcatcca ggcgccccct ggcttcgggt tctatgtaaa ctccttcatg cgccgctgcc 30540 ctgataacat ccaccaccgc agaataagcc acacccagcc aacctacaca ttcgttctgc 30600 gagtcacaca cgggaggagc gggaagagct ggaagaacca tgtttttttt ttttattcca 30660 aaagattatc caaaacctca aaatgaagat ctattaagtg aacgcgctcc cctccggtgg 30720 cgtggtcaaa ctctacagcc aaagaacaga taatggcatt tgtaagatgt tgcacaatgg 30780 cttccaaaag gcaaactgcc ctcacgtcca agtggacgta aaggctaaac ccttcanggt 30840 gaatctcctc tataaacatt ccagcacctt caaccatgcc caaataattt tcatctcgcc 30900 accttatcaa tatgtctcta agcaaatccc gaatattaag tccggccatt gtaaaaatct 30960 gctccagagc gccctccacc ttcagcctca agcagcgaat catgattgca aaaattcagg 31020 ttcctcacag acctgtataa gattcaaaag cggaacatta acaaaaatac cgcgatcccg 31080 taggtccctt cgcagggcca gctgaacata atcgtgcagg tctgcacgga ccagcgcggc 31140 cacttccccg ccaggaacca tgacaaaaga acccacactg attatgacac gcatactcgg 31200 agctatgcta accagcgtag ccccgatgta agcttgttgc atgggcggcg atataaaatg 31260 caaggtactg ctcaaaaaat caggcaaagc ctcgcgcaaa aaagcaagca catcgtagtc 31320 atgctcatgc agataaaggc aggtaagttc cggaaccacc acagaaaaag acaccatttt 31380 tctctcaaac atgtctgcgg gttcctgcat aaacacaaaa taaaataaca aaaaaaaaaa 31440 aacatttaaa cattagaagc ctgtcttaca acaggaaaaa caacccttat aagcataaga 31500 cggactacgg ccatgccggc gtgaccgtaa aaaaactggt caccgtgatt aaaaagcacc 31560 accgacagtt cctcggtcat gtccggagtc ataatgtaag actcggtaaa cacatcaggt 31620 tggttaacat cggtcagtgc taaaaagcga ccgaaatagc ccgggggaat acatacccgc 31680 aggcgtagag acaacattac agcccccata ggaggtataa caaaattaat aggagagaaa 31740 aacacataaa cacctgaaaa accctcctgc ctaggcaaaa tagcaccctc ccgctccaga 31800 acaacataca gcgcttccac agcggcagcc ataacagtca gccttaccag taaaaaaacc 31860 tattaaaaaa caccactcga cacggcacca gctcaatcag tcacagtgta aaaagggcca 31920 agtacagagc gagtatatat aggactaaaa aatgacgtaa cggttaaagt ccacaaaaac 31980 cacccagaaa accgcacgcg aacctacgcc cagaaacgaa agccaaaaaa cccacaactt 32040 cctcaaatct tcacttccgt tttcccacga tacgtcactt cccattttaa aaaaaaacta 32100 caattcccaa tacatgcaag ttactccgcc ctaaaaccta cgtcacccgc cccgttccca 32160 cgccccgcgc cacgtcacaa actccacccc ctcattatca tattggcttc aatccaaaat 32220 aaggtatatt attgatgatg aaattctaga aatcgataag cttgagtatt ctatagtgtc 32280 acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 32340 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 32400 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 32460 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 32520 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 32580 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 32640 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 32700 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 32760 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 32820 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 32880 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 32940 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 33000 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 33060 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 33120 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 33180 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 33240 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 33300 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 33360 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 33420 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 33480 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 33540 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 33600 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 33660 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 33720 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 33780 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 33840 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 33900 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 33960 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 34020 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 34080 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 34140 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 34200 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 34260 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 34320 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 34380 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 34440 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 34500 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 34560 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 34620 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 34680 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 34740 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt 34800 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta 34860 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 34920 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 34980 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 35040 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 35100 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 35160 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 35220 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 35280 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 35340 ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactata 35399 <210> 3 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> Restriction enzyme site <400> 3 ggccaagtcg gccttaatta acttaaggtt taaacgatat catttaaatt acgtactcga 60 gggatccaag ctt 73 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atgcttaatt aaatcatcaa taatatacct tattttggat tg 42 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgccttaag tttcagtccc ggtgtcggag cggctcggag 40 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atttaattaa gcgatcgcat catcaataat ataccttatt ttggattg 48 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atgcttccat caaacgagtt ggcg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tcttacctct ggtttccatg agcc 24 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atttaattaa gcgatcgcca tcatcaataa tataccttat tttgg 45 <210> 10 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Restriction enzyme site <400> 10 cttaaggagc tcttcgaact cgaggtttaa acgatatctt aattaaggat ccatttaaat 60 tctagaatcg ataagctt 78 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atcatcaata atatacctta ttttgg 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tccggctcgt atgttgtgtg gaattg 26 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tagaatcgat aagcttgagt attct 25 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 atgccttaag aattcgccct atagtgagtc gtat 34 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ctgggatttt ttaattaagt atatgag 27 <210> 16 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tactcaagct tatcgcatca tcaataatat accttatttt g 41

Claims (20)

In the serotype 6 adenovirus genome, a LITR (left Ad6 inverted terminal repeat), a nucleic acid encapsidation signal, a right Ad6 inverted terminal repeat (RITR) having a sequence of 2,939 to 2,998 in the nucleotide sequence of SEQ ID NO: 1, a right a shuttle vector for serotype 6 recombinant adenovirus comprising the arm homology and the left arm homology adjacent to the right arm homology, but not including the E1 region of the serotype 6 adenovirus genome, and a replication origin and an antibiotic resistance gene.
2. The shuttle vector according to claim 1, wherein the shuttle vector has a single restriction enzyme cleavage site at a right arm homology and a left arm homology junction.
2. The shuttle vector of claim 1, further comprising a multiple cloning site.
The shuttle vector according to claim 1, further comprising a gene encoding a target protein.
The shuttle vector according to claim 1, wherein the origin of replication and the antibiotic resistance gene are capable of being removed by restriction enzymes.
delete 2. The shuttle vector according to claim 1, wherein the RITR (right Ad6 inverted terminal repeat) has a sequence of 6,946 to 7,005 of the nucleotide sequence of SEQ ID NO: 1.
The shuttle vector according to claim 1, wherein the nucleic acid encapsidation signal has a sequence of 3,120 to 3,269 of the nucleotide sequence of SEQ ID NO: 1.
2. The method according to claim 1, wherein the right arm homology has a sequence of 3,604 to 5,848 of the nucleotide sequence of SEQ ID NO: 1 and the left arm homology has a sequence of 5,855 to 7,006 of the nucleotide sequence of SEQ ID NO: vector.
The serotype 6 adenovirus genome from which the E1 region has been deleted, having a sequence of 3,604 to 5,848 of the nucleotide sequence of SEQ ID NO: 1 right arm homology and the right arm homology, and a left arm homology having a sequence of 5,855 to 7,006 in the nucleotide sequence of SEQ ID NO: 1, a replication origin, and a rescue vector for producing serotype 6 recombinant adenovirus comprising an antibiotic resistance gene vector).
[Claim 11] The rescue vector for preparing serotype 6 recombinant adenovirus according to claim 10, wherein the deleted El region is a LITR to AF11 region.
The recombinant adenoviral vector according to claim 10, wherein the serotype 6 adenovirus genome from which the E1 region has been deleted has a nucleotide sequence of 10 to 32,251 of the nucleotide sequence of SEQ ID NO: 2. ).
delete A method for preparing a recombinant serotype 6 adenovirus vector comprising the steps of:
(a) transfecting the shuttle vector of any one of claims 1 to 5 and 7 to 9 and the rescue vector of any one of claims 10 to 12 into a microorganism having recombinase activity Homologous recombination;
(b) culturing the transformed microorganism; And
(c) obtaining a recombinant serotype 6 adenovirus vector from the cultured microorganism.
15. The method according to claim 14, wherein the shuttle vector is linearized by digesting a right arm homology and a left arm homology linkage region with restriction enzymes before transformation.
15. The method for producing a recombinant serotype 6 adenoviral vector according to claim 14, wherein the microorganism having recombinase activity is an E. coli or adenovirus producing cell line.
A recombinant serotype 6 adenovirus vector, produced by the method of claim 14, which contains serotype 6 adenovirus genome, promoter, origin of replication and antibiotic resistance gene which does not contain the E1 gene.
The recombinant serotype 6 adenovirus vector according to claim 17, further comprising a gene encoding a target protein.
19. The recombinant serotype 6 adenovirus vector according to claim 18, wherein the target protein is a therapeutic protein.
delete

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