CN109576269B - Animal cell expression vector containing MAR core fragment - Google Patents

Abstract

The invention discloses an animal cell expression vector containing a MAR core fragment. The nucleotide base sequence of the MAR core fragment of the recombinant expression vector is shown as SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, respectively. The expression vector of the invention can effectively improve the yield of protein in mammalian cells and reduce the production cost.

Description

Animal cell expression vector containing MAR core fragment

The present application is a divisional application of 'a novel MAR core fragment-containing animal cell expression vector' invention patent application No. 201410129979.2 filed 4/1/2014.

Technical Field

The invention relates to the technical field of biology, and particularly discloses an animal cell expression vector containing a MAR core fragment.

Background

At present, many vectors have been developed for producing recombinant proteins that can be expressed in high yield in bacterial, eukaryotic microorganisms, insect cells, etc., but these expression systems lack protein modification mechanisms (e.g., glycosylation modification, etc.) similar to those of mammalian cells, and the expressed animal proteins are prone to lack biological activity or to produce inclusion bodies due to folding errors. However, the production of recombinant proteins by mammalian cell expression systems is often low in yield, and the stability of the introduced gene is problematic. Later, Chinese Hamster Ovary (CHO) cells were found to be the best system for producing complex macromolecules such as monoclonal antibodies, and the yield of the monoclonal antibodies produced by the CHO expression system is greatly improved, but the selection and maintenance of high expression cell lines are still time-consuming and labor-consuming and uncertain. Following cell transfection, the fusion gene is usually randomly integrated into the genome of the host cell, with most recombination occurring in "silent regions," e.g., non-transcribed regions. Often thousands of clones need to be screened to obtain several cell lines with a sufficient yield.

The Matrix Attachment Region (MAR) element is a DNA sequence that helps create and maintain open chromatin domains. The open chromatin domains facilitate transcription, expression and multicopy integration of transgenes. The MAR element is put into the vector to increase the cell line yield and the proportion of high-producing cell lines (Kwaks TH, Otte AP2006Trends Biotechnol 24: 137-142), but the MAR element is larger (more than 3 kb), and the efficiency of plasmid transfection of cells is reduced along with the increase of plasmid size (Yin W et al Anal biochem.2005 Nov15; 346(2):289-94), thereby limiting the application of the MAR element in the double-subunit coexpression vector.

Therefore, the development of a more efficient mammalian cell expression vector has been a problem to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide an expression vector capable of improving the expression of an introduced gene in mammalian cells, wherein the two ends of the expression vector contain MAR core fragments of a nuclear matrix attachment region of an artificial transcription factor binding site. The expression vector can effectively improve the yield of the protein in mammalian cells and reduce the production cost.

Specifically, the invention discloses:

1. an isolated MAR core fragment of a recombinant expression vector, having a nucleotide base sequence as set forth in SEQ ID NO:

1. SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

2. A recombinant expression vector comprising at both ends the MAR core fragment of claim 1 above.

3. The vector of 2 above, wherein said MAR core fragment is inserted upstream of a promoter and downstream of a polyadenylation site.

4. The vector of 3 above, wherein the promoter is CMV, SV40 or EF-1a promoter. Preferably, the MAR core fragment is inserted into the vector at an FSP I site upstream of the vector, or at an Afe I site downstream of SV40PA, or at an Nhe I site upstream of PEF-1a, or at an EcoRV site downstream of BGHPA. Most preferred are the upstream FSP I site and the upstream Nhe I site of PEF-1 a.

5. The vector of any one of the above 2-4, wherein the vector is pBudCE4.1, pDR1, pcDNA3.1(+), pDFHF or pCHO1.0, or a modified vector of the above vector.

6. The vector of the above 5, which is a pBudCE4.1 modified vector, has a nucleotide sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 or SEQ ID NO: and 6.

7. The vector of any one of the above 2-6, wherein the vector comprises one or more functional recombinant genes encoding one or more recombinant proteins.

8. A host cell comprising the vector of 7 above.

9. The cell of 8 above, which is a mammalian cell, including but not limited to a cell selected from the group consisting of: chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK 293) cells, Baby Hamster Kidney (BHK) cells, Hela cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, PerC6 cells, and MDCK cells.

10. A method of producing a recombinant protein, said method comprising transcribing and translating one or more genes encoding said recombinant protein present in any one of the expression vectors described above.

The expression vector is particularly suitable for expression of mammalian cells, particularly Chinese Hamster Ovary (CHO) cells.

The MAR core fragment DNA sequence with artificial transcription factor binding sites added at two ends can be inserted into a mammalian cell expression vector, so that the expression of the introduced mammalian gene in mammalian cells is greatly improved, and the stability is good.

The invention provides a mammalian cell expression vector, which contains MAR core fragment DNA sequences with artificial transcription factor binding sites added at two ends, wherein the expression vector is a modified pBudCE4.1 vector, and Afe I and EcoR V restriction enzyme cutting sites are respectively inserted at the downstream of SV40 polyadenylation site and BGH polyadenylation site in the modified pBudCE4.1 vector. The Zeocin resistance gene in pBudCE4.1 was replaced with kanamycin resistance gene and DHFR gene. The modified vectors are named pBCM1.1, pBCM1.2 and pBCM1.3, and the DNA sequences are respectively shown in SEQ ID No.4, SEQ ID No. 5 and SEQ ID No. 6. The modified vector which does not contain the nuclear matrix binding region element fragment and the artificial transcription factor binding site DNA is named pBCM1.0, and the DNA sequence is shown as SEQ ID No 7.

The MAR core fragment DNA sequence with artificial transcription factor binding sites added at both ends can be prepared by a conventional synthetic method.

The plasmids of the present invention containing MAR core fragments with artificial transcription factor binding sites at both ends were compared with plasmids containing no MAR core fragments with artificial transcription factor binding sites at both ends. The results of the determination of the expression level of the foreign protein in the two plasmids show that the plasmid of the MAR core fragment with the artificial transcription factor binding sites added at the two ends is particularly suitable for CHO cells, and compared with the plasmid of the MAR core fragment without the nuclear matrix binding region elements with the artificial transcription factor binding sites added at the two ends, the expression level of the foreign protein in animal cells is greatly improved, and the expression level is improved by 10 to 17 times. The proportion of high-yield cell strains is increased by 25-40 times.

The gene of the foreign protein (e.g., therapeutic protein, antibody) is inserted into the expression vector of the present invention, and then transfected into mammalian cells, which can be used for the production of the foreign protein (e.g., therapeutic protein, antibody).

Drawings

FIG. 1 is a graph comparing the average GFP expression levels of expression vectors containing the MAR core fragment of the present invention with expression vectors containing no MAR gene or MAR full-length gene sequences.

FIG. 2 is a graph comparing the ratio of mean high expression cell lines for GFP from expression vectors containing the MAR core fragment of the invention to expression vectors containing no MAR gene or full-length MAR gene sequences.

FIG. 3 is a graph showing the comparison of the average CAT expression levels of expression vectors containing the MAR core fragment of the present invention and expression vectors containing no MAR gene or containing the MAR full-length gene sequence.

FIG. 4 is a graph comparing the ratio of CAT average high expressing cell lines of expression vectors containing the MAR core fragment of the present invention to expression vectors containing no MAR gene or MAR full length gene sequences.

FIG. 5 Structure of pBCM1.0 plasmid.

Detailed Description

The following examples and experimental examples are intended to further illustrate the present invention and should not be construed as limiting the present invention.

The expression vectors listed in the examples of the present invention were obtained by modifying pBudCE4.1 vector (available from Life technologies): respectively inserting AfeI restriction enzyme cutting sites and EcoR V restriction enzyme cutting sites at the downstream of SV40 polyadenylation sites and BGH polyadenylation sites; the Kanamycin Resistance Gene (Kanamycin Resistance Gene), PGK promoter, dihydrofolate reductase (DHFR), and SV40PA were PCR-derived from pCHO1.0 (from Life Technologies) and loaded into pBudCE4.1 through NheI and AfeI sites, with the removal of the Zeocin Resistance Gene; the Chloramphenicol acetyl transferase gene (CAT gene) was PCR-out from pBudCE4.1/lacZ/CAT (from Life Technologies) and loaded into pBudCE4.1 through Kpn I and Bgl II sites; the Green Fluorescent Protein gene (GFP gene) was PCR-out from PCDNA3.1/CT-GFP-TOP9 (from Life technologies) and loaded into pBudCE4.1 through Pst I and BamH I sites.

Example 1 introduction of Afe I and EcoR V restriction sites into pBudCE4.1

The Afe I and EcoR V restriction enzyme cutting sites are introduced into the downstream of SV40 polyadenylation site and BGH polyadenylation site by a point mutation method. Point mutations were performed using the Quickchange Point mutation kit (from Agilent technologies). Introducing an Afe I enzyme cutting site.

A forward primer: 5'-GGAAAACGATTCCGAAGC GCT AC-3'

Reverse primer: 5'-CCT TCT ATG AAA GGT AGC GCT TCG-3'

The site-directed mutagenesis PCR amplification conditions are as follows: 1. 30 seconds at 95 ℃; 2. 30 seconds at 95 ℃; 3.1 minute at 55 ℃; 4. 5 minutes at 68 ℃; 5. 2-4 repeat 18 cycles.

The template plasmid was digested by addition of Dpn I enzyme and then transformed into XL1-Blue competent E.coli. The method comprises the following specific steps: 1. freshly prepared or stored at-20 ℃ 100uL of competent cells (purchased from Agilent technologies) were placed on ice and after complete deicer, the cells were gently suspended evenly. 2. The digested plasmid was added with Dpn I enzyme (from NEB) and gently mixed. 3. The mixture was left on ice for 30 minutes. The heat shock in the water bath was carried out at 4.42 ℃ for 60 seconds. 5. The mixture was left on ice for 2 minutes. 6. 400uL of LB medium (purchased from Sigma-Aldrich) was added and incubated at 37 ℃ for 30 minutes with shaking at 250 rpm. 7. The bacteria were spread on Amp/LB agar plates 8. the plates were placed in the forward direction at 37 ℃ for 1 hour, after the inoculated liquid had absorbed into the agar, the plates were inverted and incubated overnight. 9. Colonies which appeared on ampicillin/LB agar plates after overnight culture at 37 ℃ were plasmid-transformed E.coli, 10, monoclonal bacteria were selected and plasmids were extracted. After obtaining the mutant plasmid, the mutant plasmid is cut by Afe I enzyme for identification and sent to Shanghai Bioengineering Co., Ltd for sequencing, and the result is correct.

Then introducing an EcoR V enzyme cutting site into the vector into which the AfeI has been introduced:

a forward primer: 5'-ATCCACAGAATCAGGGGATAT CGC-3'

Reverse primer: 5'-CAC ATG TTC TTT CCT GCG ATA TCC C-3'

The site-directed mutagenesis PCR amplification conditions are as follows: 1. 95 ℃ for 30 seconds. 2. 30 seconds at 95 ℃; 3.1 minute at 55 ℃; 4. 5 minutes at 68 ℃; 5. 2-4 repeat 18 cycles

The template plasmid was digested by addition of Dpn I enzyme and then transformed into XL1-Blue competent E.coli. After obtaining the mutant plasmid, the EcoR V enzyme digestion identification is correct, and the sequencing is sent to Shanghai biological engineering company Limited for sequencing, and the result is correct.

Example 2 pBudCE4.1 in which the Zeocin resistance gene was deleted and the kanamycin resistance gene, PGK promoter, DHFR and SV40PA were added

The kanamycin resistance gene, PGK promoter, DHFR and SV40PA were PCR-derived from pCHO1.0 (from Life technologies) and loaded into pBudCE4.1 obtained in example 1 via NheI and AfeI sites, with the removal of the Zeocin resistance gene.

Forward primer 5'-GTG AGCGCTTTAGAAAAACTCATCGAGCATC-3'

Reverse primer: 5'-GTG GCTAGCTAA GAT ACA TTG ATG AGT TTG-3'

Primestar HS high fidelity DNA polymerase (from TAKARA) was used for PCR,

the method comprises the following specific steps: 1.3 minutes at 95 ℃; 2. 10 seconds at 98 ℃; 3. 15 seconds at 47 ℃; 4. 3 minutes at 72 ℃; 5. 2-4 repeating 30 cycles; 6. 5 minutes at 72 ℃.

The structure diagram of the modified new vector is shown in figure 5, wherein, the NheI, AfeI, FspI and EcoRV sites are insertion sites of MAR core fragments with artificial transcription factor binding sites added at both ends.

Example 3 construction of expression vector containing MAR core fragment flanked by binding sites for an Artificial transcription factor

MAR core fragments (with sequences shown as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) with artificial transcription factor binding sites added at both ends were synthesized by Shanghai Bioengineering Co., Ltd.

MAR core fragments with artificial transcription factor binding sites added at both ends can be inserted into the FSP I site upstream of PBCM1.0, or the Afe I site downstream of SV40PA, or the Nhe I site upstream of PEF-1a, or the EcoRV site downstream of BGH PA.

In the following examples, MAR core fragments with artificial transcription factor binding sites added at both ends were inserted at FSP I site upstream of PBCM1.0 and Nhe I site upstream of PEF-1a, and the sequences of MAR core fragments are shown in SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

The MAR core fragment with artificial transcription factor binding sites added at both ends is ligated to the PBCM1.0 upstream FSP I site by blunt end. And the sequencing is correct.

The MAR core fragment with artificial transcription factor binding sites added at both ends was ligated to the PBCM1.0 upstream Nhe I site by sticky ends. And the sequencing is correct.

The constructed expression vectors of MAR core fragments with artificial transcription factor binding sites added at both ends are named pBCM1.1, pBCM1.2 and pBCM1.3 respectively, and the DNA sequences are SEQ ID Nos. 4, 5 and 6 respectively.

Example 4 insertion of foreign protein Gene into expression vector containing MAR core fragment having Artificial transcription factor binding sites added at both ends

CAT gene inserted into KpnI and BglII site downstream of PEF-1a

The CAT gene was PCR-extracted from pBudCE4.1/lacZ/CAT (Life technologies) and loaded into the expression vector constructed in example 3 above through Kpn I and Bgl II sites.

Forward primer 5'-GTG GGTACC ATGGAGAAAAAAATCACTGG-3'

Reverse primer: 5'-GTG AGATCT TTA CGCCCCGCCCTGCCACTCATC-3'

Primestar HS high fidelity DNA polymerase (from TAKARA) was used for PCR,

the method comprises the following specific steps: 3 minutes at 95 ℃; 2. 10 seconds at 98 ℃; 3. 15 seconds at 52 ℃; 4.1 minute at 72 ℃ and 30 cycles repeated 2-4; 5. 5 minutes at 72 DEG C

The expression level of CAT gene was determined using chloramphenicol acetyl transferase assay kit (purchased from Life Technologies).

The GFP gene was PCR-derived from PCDNA3.1/CT-GFP-TOP9 (available from Life Technologies) and loaded into the expression vector constructed in example 3 via the downstream Pst I, BamH I site.

Forward primer 1: 5'-GTG CTGCAT GCCACCATGGCTAGCAAAGGAGAAG-3'

Reverse primer 1: 5'-GAGGGATCC TCATTATTTGTAGAGCTCATC-3'

Primestar HS high fidelity DNA polymerase (from TAKARA) was used for PCR,

the method comprises the following specific steps: 3 minutes at 95 ℃; 2. 10 seconds at 98 ℃; 3. 15 seconds at 50 ℃; 4.1 minute at 72 ℃ and 30 cycles repeated 2-4; 5. 5 minutes at 72 DEG C

The expression level of GFP can be determined by cytofluorimetric analyses (BD FACSCalibur flowcytometer) (from BD Co.) and analyzed by WinMDI 2.8 software.

Example 5 expression of GFP and CAT vectors containing MAR core fragments flanked by binding sites for Artificial transcription factors

The effect of MAR core fragment (SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) with artificial transcription factor binding sites added at both ends on increasing the expression level of green fluorescent protein and chloramphenicol acetyltransferase in transfected CHO cells.

The seeds were transfected using FreeStyle MAX transfection reagent (from Life Technologies)The expression vectors pBCM1.1, pBCM1.2, pBCM1.3 constructed in example 4 were introduced into CHO-S cells (purchased from Life Technologies). The transfected cells were placed at 37 ℃ in 8% CO₂130 ℃ and 150 rpm. Stable expression screening was started after 48 hours. Expression vectors containing no MAR and full-length MAR-containing sequences were also used as controls. The results of the experiments are shown in FIGS. 1-4, and the results show that the expression vector containing the MAR core fragment of the present invention has greatly increased the expression level of the foreign protein in animal cells, and the expression level of GFP has been increased by 10-17 times (see FIG. 1). CAT expression was increased 14-15 fold (see FIG. 3). The proportion of GFP producing cell lines was increased by 25-40 fold (see FIG. 2). The proportion of CAT high-producing cell lines is increased by 25-40 times (see figure 4).

Sequence listing

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<120> a novel MAR core fragment-containing animal cell expression vector

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gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag 1980

acacaacgtg gctttccccc cccccccatt attgaagcat ttatcagggt tattgtctca 2040

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 2100

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 2160

aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 2220

tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 2280

gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 2340

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 2400

gcgtaaggag aaaataccgc atcagattgg ctattggcca ttgcatacgt tgtatccata 2460

tcataatatg tacaattcgc ccttcattta attctaccgg gtaggggagg cgcttttccc 2520

aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 2580

cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 2640

tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 2700

gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 2760

ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 2820

ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 2880

gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 2940

gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttctcg 3000

agctcaagct tggggggggg gacagctcag ggctgcgatt tcgcgccaaa cttgacggca 3060

atcctagcgt gaaggctggt aggattttat ccccgctgcc atcatggttc gaccattgac 3120

tgcatcgtcg ccgtgtccca aaatatgggg attggcaaga acggagacct accctggcct 3180

ccgctcagga acgagttcaa gtacttccaa agaatgacca caacctcttc agtggaaggt 3240

aaacagaatc tggtgattat gggtaggaaa acctggttct ccattcctga gaagaatcga 3300

cctttaaagg acagaattaa tatagttctc agtagagaac tcaaagaacc accacgagga 3360

gctcattttc ttgccaaaag tttggatgat gccttaagac ttattgaaca accggaattg 3420

gcaagtaaag tagacatggt ttggatagtc ggaggcagtt ctgtttacca ggaagccatg 3480

aatcaaccaggccacctcag actctttgtg acaaggatca tgcaggaatt tgaaagtgac 3540

acgtttttcc cagaaattga tttggggaaa tataaacttc tcccagaata cccaggcgtc 3600

ctctctgagg tccaggagga aaaaggcatc aagtataagt ttgaagtcta cgagaagaaa 3660

gactaagcta gctctttaat tctaatatat ttagaattca tctttaattc taatatattt 3720

agaattcttc tttaattcta atatatttag aattcctctt taattctaat atatttagaa 3780

ttcttcttta attctaatat atttagaatt cctctttaat tctaatatat ttagaattca 3840

tctttaattc taatatattt agaattcctc tttaattcta atatatttag aattcttctt 3900

taattctaat atatttagaa ttcatatata tacacatata tataaaatat atatacacac 3960

atatatataa aatatatata tacacacata tatataaaat atatatatac acatatatat 4020

aaaatatata tatacacata tatataaaat atatatatat acacatatat ataaaatata 4080

tatacacaca tatatataaa gtatatatat acacacatat atataaaata tatatataca 4140

catatatata aaatatatat atacacatat atataaaata tatatataca catatatata 4200

aaaatatata tatatatttt ttaaaatatt ccaattgtct cactttgtgg atgagaaaaa 4260

gaagtagtta gaggtcaagt aacttggcct acatcttttc tcaagattgt aaactcctag 4320

tgagcaataa ccacatcttc attttctttg tataaaacaa gaaagtttag catgaaaaag 4380

gtactcaatt acaaatgtgt tggattgaat tgaagaccct tggaagggga ttttgtacct 4440

gaggatctct ttcttttggc catattgttc aatggacaaa atttagcctt cgaaggcagg 4500

ccgatttgag gttaatacta cctttaccac ttgatagcta tgtgaccttg gccatgtggt 4560

ttcaacagtc tgaacctcat tttctctgtg tatgtgtggt cctccttaca agtttgtgaa 4620

aaatgtgaag tccttagcca tgatagccca atataacagg ctaaatgata ataggtttat 4680

gttcttttcc tttatattct cagataagca ctgtcctctt taattctaat atatttagaa 4740

ttcatcttta attctaatat atttagaatt cttctttaat tctaatatat ttagaattcc 4800

tctttaattc taatatattt agaattcttc tttaattcta atatatttag aattcctctt 4860

taattctaat atatttagaa ttcatcttta attctaatat atttagaatt cctctttaat 4920

tctaatatat ttagaattct tctttaattc taatatattt agaattcagc tagctgaggc 4980

tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga 5040

ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat 5100

gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta 5160

gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg 5220

tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact 5280

tccacctggc tccagtacgt gattcttgat cccgagctgg agccaggggc gggccttgcg 5340

ctttaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc 5400

gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca 5460

tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg 5520

cgggccagga tctgcacact ggtatttcgg tttttgggcc cgcggccggc gacggggccc 5580

gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg 5640

gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta 5700

tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat 5760

ggccgcttcc cggccctgct ccagggggct caaaatggag gacgcggcgc tcgggagagc 5820

gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat 5880

gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctgg agcttttgga 5940

gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt 6000

gggtggagac tgaagttagg ccagcttggc acttgatgta attctcgttg gaatttgccc 6060

tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc 6120

ttccatttca ggtgtcgtga acacgtggtc gcggccgctt cgaaggtacc atggagaaaa 6180

aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg 6240

catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct 6300

ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg 6360

cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga 6420

tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat 6480

cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg 6540

tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt 6600

tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg 6660

acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc 6720

tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa 6780

tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa agatctggcc 6840

ggctgggccc gtttcgaagg taagcctatc cctaaccctc tcctcggtct cgattctacg 6900

cgtaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg 6960

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 7020

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 7080

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 7140

gacaatagca ggcatgctgg ggatgcggtg ggctctatgg atatcgcttc tgaggcggaa 7200

agaaccagtg gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 7260

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7320

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7380

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7440

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7500

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7560

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7620

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7680

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7740

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7800

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt7860

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7920

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7980

gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga 8040

tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 8100

ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 8160

ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt 8220

gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg 8280

ctgcgcatct ttaattctaa tatatttaga attcatcttt aattctaata tatttagaat 8340

tcttctttaa ttctaatata tttagaattc ctctttaatt ctaatatatt tagaattctt 8400

ctttaattct aatatattta gaattcctct ttaattctaa tatatttaga attcatcttt 8460

aattctaata tatttagaat tcctctttaa ttctaatata tttagaattc ttctttaatt 8520

ctaatatatt tagaattcat atatatacac atatatataa aatatatata cacacatata 8580

tataaaatat atatatacac acatatatat aaaatatata tatacacata tatataaaat 8640

atatatatac acatatatat aaaatatata tatatacaca tatatataaa atatatatac 8700

acacatatat ataaagtata tatatacaca catatatata aaatatatat atacacatat 8760

atataaaata tatatataca catatatata aaatatatat atacacatat atataaaaat 8820

atatatatat attttttaaa atattccaat tgtctcactt tgtggatgag aaaaagaagt 8880

agttagaggt caagtaactt ggcctacatc ttttctcaag attgtaaact cctagtgagc 8940

aataaccaca tcttcatttt ctttgtataa aacaagaaag tttagcatga aaaaggtact 9000

caattacaaa tgtgttggat tgaattgaag acccttggaa ggggattttg tacctgagga 9060

tctctttctt ttggccatat tgttcaatgg acaaaattta gccttcgaag gcaggccgat 9120

ttgaggttaa tactaccttt accacttgat agctatgtga ccttggccat gtggtttcaa 9180

cagtctgaac ctcattttct ctgtgtatgt gtggtcctcc ttacaagttt gtgaaaaatg 9240

tgaagtcctt agccatgata gcccaatata acaggctaaa tgataatagg tttatgttct 9300

tttcctttat attctcagat aagcactgtc ctctttaatt ctaatatatt tagaattcat 9360

ctttaattct aatatattta gaattcttct ttaattctaa tatatttaga attcctcttt 9420

aattctaata tatttagaat tcttctttaa ttctaatata tttagaattc ctctttaatt 9480

ctaatatatt tagaattcat ctttaattct aatatattta gaattcctct ttaattctaa 9540

tatatttaga attcttcttt aattctaata tatttagaat tcatgcgcaa ctgttgggaa 9600

gggcgatcgg tgcgggcctc ttcgctatta cgcca 9635

<210>5

<211>9995

<212>DNA

<213>Synthesized DNA

<400>5

gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180

caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360

tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420

gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480

gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600

taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660

cccaagcttg cattcctgca ggtcgacatc gatcttaagc agtacttcta gaggatccga 720

acaaaaactc atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca 780

ttgagtttga tccccgggaa ttcagacatg ataagataca ttgatgagtt tggacaaacc 840

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 900

tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag 960

atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct 1020

ttcatagaag gcggcggtgg aatcgaaatc tcgtagcacg tgtagcgctt tagaaaaact 1080

catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 1140

gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 1200

gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 1260

cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 1320

agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct 1380

cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 1440

gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 1500

gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 1560

cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 1620

ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 1680

tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 1740

catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 1800

cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgagcaag 1860

acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca 1920

gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag 1980

acacaacgtg gctttccccc cccccccatt attgaagcat ttatcagggt tattgtctca 2040

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 2100

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 2160

aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 2220

tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 2280

gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 2340

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 2400

gcgtaaggag aaaataccgc atcagattgg ctattggcca ttgcatacgt tgtatccata 2460

tcataatatg tacaattcgc ccttcattta attctaccgg gtaggggagg cgcttttccc 2520

aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 2580

cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 2640

tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 2700

gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 2760

ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 2820

ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 2880

gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 2940

gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttctcg 3000

agctcaagct tggggggggg gacagctcag ggctgcgatt tcgcgccaaa cttgacggca 3060

atcctagcgt gaaggctggt aggattttat ccccgctgcc atcatggttc gaccattgac 3120

tgcatcgtcg ccgtgtccca aaatatgggg attggcaaga acggagacct accctggcct 3180

ccgctcagga acgagttcaa gtacttccaa agaatgacca caacctcttc agtggaaggt 3240

aaacagaatc tggtgattat gggtaggaaa acctggttct ccattcctga gaagaatcga 3300

cctttaaagg acagaattaa tatagttctc agtagagaac tcaaagaacc accacgagga 3360

gctcattttc ttgccaaaag tttggatgat gccttaagac ttattgaaca accggaattg 3420

gcaagtaaag tagacatggt ttggatagtc ggaggcagtt ctgtttacca ggaagccatg 3480

aatcaaccag gccacctcag actctttgtg acaaggatca tgcaggaatt tgaaagtgac 3540

acgtttttcc cagaaattga tttggggaaa tataaacttc tcccagaata cccaggcgtc 3600

ctctctgagg tccaggagga aaaaggcatc aagtataagt ttgaagtcta cgagaagaaa 3660

gactaagcta gcgatcagtt gcgtaatcta tgctgcattg cgcaattcta gatcagttgc 3720

gtaatctatg ctgcattgcg caattcttga tcagttgcgt aatctatgct gcattgcgca 3780

attctcgatc agttgcgtaa tctatgctgc attgcgcaat tcttgatcag ttgcgtaatc 3840

tatgctgcat tgcgcaattc tcgatcagtt gcgtaatcta tgctgcattg cgcaattcta 3900

gatcagttgc gtaatctatg ctgcattgcg caattctcga tcagttgcgt aatctatgct 3960

gcattgcgca attcttgatc agttgcgtaa tctatgctgc attgcgcaat tctatatata 4020

tacacatata tataaaatat atatacacac atatatataa aatatatata tacacacata 4080

tatataaaat atatatatac acatatatat aaaatatata tatacacata tatataaaat 4140

atatatatat acacatatat ataaaatata tatacacaca tatatataaa gtatatatat 4200

acacacatat atataaaata tatatataca catatatata aaatatatat atacacatat 4260

atataaaata tatatataca catatatata aaaatatata tatatatttt ttaaaatatt 4320

ccaattgtct cactttgtgg atgagaaaaa gaagtagtta gaggtcaagt aacttggcct 4380

acatcttttc tcaagattgt aaactcctag tgagcaataa ccacatcttc attttctttg 4440

tataaaacaa gaaagtttag catgaaaaag gtactcaatt acaaatgtgt tggattgaat 4500

tgaagaccct tggaagggga ttttgtacct gaggatctct ttcttttggc catattgttc 4560

aatggacaaa atttagcctt cgaaggcagg ccgatttgag gttaatacta cctttaccac 4620

ttgatagcta tgtgaccttg gccatgtggt ttcaacagtc tgaacctcat tttctctgtg 4680

tatgtgtggt cctccttaca agtttgtgaa aaatgtgaag tccttagcca tgatagccca 4740

atataacagg ctaaatgata ataggtttat gttcttttcc tttatattct cagataagca 4800

ctgtccgatc agttgcgtaa tctatgctgc attgcgcaat tctagatcag ttgcgtaatc 4860

tatgctgcat tgcgcaattc ttgatcagtt gcgtaatcta tgctgcattg cgcaattctc 4920

gatcagttgc gtaatctatg ctgcattgcg caattcttga tcagttgcgt aatctatgct 4980

gcattgcgca attctcgatc agttgcgtaa tctatgctgc attgcgcaat tctagatcag 5040

ttgcgtaatc tatgctgcat tgcgcaattc tcgatcagtt gcgtaatcta tgctgcattg 5100

cgcaattctt gatcagttgc gtaatctatg ctgcattgcg caattctagc tagctgaggc 5160

tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga 5220

ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat 5280

gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta 5340

gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg 5400

tgtggttccc gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact 5460

tccacctggc tccagtacgt gattcttgat cccgagctgg agccaggggc gggccttgcg 5520

ctttaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc 5580

gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca 5640

tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg 5700

cgggccagga tctgcacact ggtatttcgg tttttgggcc cgcggccggc gacggggccc 5760

gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg 5820

gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta 5880

tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat 5940

ggccgcttcc cggccctgct ccagggggct caaaatggag gacgcggcgc tcgggagagc 6000

gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat 6060

gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctgg agcttttgga 6120

gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt 6180

gggtggagac tgaagttagg ccagcttggc acttgatgta attctcgttg gaatttgccc 6240

tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc 6300

ttccatttca ggtgtcgtga acacgtggtc gcggccgctt cgaaggtacc atggagaaaa 6360

aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg 6420

catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct 6480

ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg 6540

cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga 6600

tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat 6660

cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg 6720

tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt 6780

tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg 6840

acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc 6900

tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa 6960

tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa agatctggcc 7020

ggctgggccc gtttcgaagg taagcctatc cctaaccctc tcctcggtct cgattctacg 7080

cgtaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg 7140

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 7200

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 7260

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 7320

gacaatagca ggcatgctgg ggatgcggtg ggctctatgg atatcgcttc tgaggcggaa 7380

agaaccagtg gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 7440

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7500

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7560

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7620

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7680

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7740

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7800

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7860

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7920

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7980

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 8040

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 8100

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 8160

gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga 8220

tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 8280

ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 8340

ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt 8400

gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg 8460

ctgcgcagat cagttgcgta atctatgctg cattgcgcaa ttctagatca gttgcgtaat 8520

ctatgctgca ttgcgcaatt cttgatcagt tgcgtaatct atgctgcatt gcgcaattct 8580

cgatcagttg cgtaatctat gctgcattgc gcaattcttg atcagttgcg taatctatgc 8640

tgcattgcgc aattctcgat cagttgcgta atctatgctg cattgcgcaa ttctagatca 8700

gttgcgtaat ctatgctgca ttgcgcaatt ctcgatcagt tgcgtaatct atgctgcatt 8760

gcgcaattct tgatcagttg cgtaatctat gctgcattgc gcaattctat atatatacac 8820

atatatataa aatatatata cacacatata tataaaatat atatatacac acatatatat 8880

aaaatatata tatacacata tatataaaat atatatatac acatatatat aaaatatata 8940

tatatacaca tatatataaa atatatatac acacatatat ataaagtata tatatacaca 9000

catatatata aaatatatat atacacatat atataaaata tatatataca catatatata 9060

aaatatatat atacacatat atataaaaat atatatatat attttttaaa atattccaat 9120

tgtctcactt tgtggatgag aaaaagaagt agttagaggt caagtaactt ggcctacatc 9180

ttttctcaag attgtaaact cctagtgagc aataaccaca tcttcatttt ctttgtataa 9240

aacaagaaag tttagcatga aaaaggtact caattacaaa tgtgttggat tgaattgaag 9300

acccttggaa ggggattttg tacctgagga tctctttctt ttggccatat tgttcaatgg 9360

acaaaattta gccttcgaag gcaggccgat ttgaggttaa tactaccttt accacttgat 9420

agctatgtga ccttggccat gtggtttcaa cagtctgaac ctcattttct ctgtgtatgt 9480

gtggtcctcc ttacaagttt gtgaaaaatg tgaagtcctt agccatgata gcccaatata 9540

acaggctaaa tgataatagg tttatgttct tttcctttat attctcagat aagcactgtc 9600

cgatcagttg cgtaatctat gctgcattgc gcaattctag atcagttgcg taatctatgc 9660

tgcattgcgc aattcttgat cagttgcgta atctatgctg cattgcgcaa ttctcgatca 9720

gttgcgtaat ctatgctgca ttgcgcaatt cttgatcagt tgcgtaatct atgctgcatt 9780

gcgcaattct cgatcagttg cgtaatctat gctgcattgc gcaattctag atcagttgcg 9840

taatctatgc tgcattgcgc aattctcgat cagttgcgta atctatgctg cattgcgcaa 9900

ttcttgatca gttgcgtaat ctatgctgca ttgcgcaatt ctatgcgcaa ctgttgggaa 9960

gggcgatcgg tgcgggcctc ttcgctatta cgcca 9995

<210>6

<211>9947

<212>DNA

<213>Synthesized DNA

<400>6

gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180

caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360

tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420

gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480

gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600

taactagaga acccactgct tactggctta tcgaaattaa tacgactcac tatagggaga 660

cccaagcttg cattcctgca ggtcgacatc gatcttaagc agtacttcta gaggatccga 720

acaaaaactc atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca 780

ttgagtttga tccccgggaa ttcagacatg ataagataca ttgatgagtt tggacaaacc 840

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 900

tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag 960

atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct 1020

ttcatagaag gcggcggtgg aatcgaaatc tcgtagcacg tgtagcgctt tagaaaaact 1080

catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 1140

gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 1200

gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 1260

cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 1320

agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct 1380

cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 1440

gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 1500

gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 1560

cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 1620

ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 1680

tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 1740

catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 1800

cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgagcaag 1860

acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca 1920

gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag 1980

acacaacgtg gctttccccc cccccccatt attgaagcat ttatcagggt tattgtctca 2040

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 2100

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 2160

aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 2220

tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 2280

gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 2340

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 2400

gcgtaaggag aaaataccgc atcagattgg ctattggcca ttgcatacgt tgtatccata 2460

tcataatatg tacaattcgc ccttcattta attctaccgg gtaggggagg cgcttttccc 2520

aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 2580

cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 2640

tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 2700

gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 2760

ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 2820

ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 2880

gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 2940

gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttctcg 3000

agctcaagct tggggggggg gacagctcag ggctgcgatt tcgcgccaaa cttgacggca 3060

atcctagcgt gaaggctggt aggattttat ccccgctgcc atcatggttc gaccattgac 3120

tgcatcgtcg ccgtgtccca aaatatgggg attggcaaga acggagacct accctggcct 3180

ccgctcagga acgagttcaa gtacttccaa agaatgacca caacctcttc agtggaaggt 3240

aaacagaatc tggtgattat gggtaggaaa acctggttct ccattcctga gaagaatcga 3300

cctttaaagg acagaattaa tatagttctc agtagagaac tcaaagaacc accacgagga 3360

gctcattttc ttgccaaaag tttggatgat gccttaagac ttattgaaca accggaattg 3420

gcaagtaaag tagacatggt ttggatagtc ggaggcagtt ctgtttacca ggaagccatg 3480

aatcaaccag gccacctcag actctttgtg acaaggatca tgcaggaatt tgaaagtgac 3540

acgtttttcc cagaaattga tttggggaaa tataaacttc tcccagaata cccaggcgtc 3600

ctctctgagg tccaggagga aaaaggcatc aagtataagt ttgaagtcta cgagaagaaa 3660

gactaagcta gctctttaat tctaatatat ttagaattca tctttaattc taatatattt 3720

agaattcttc tttaattcta atatatttag aattcctctt taattctaat atatttagaa 3780

ttcttcttta attctaatat atttagaatt ccgatcagtt gcgtaatcta tgctgcattg 3840

cgcaattcta gatcagttgc gtaatctatg ctgcattgcg caattcttga tcagttgcgt 3900

aatctatgct gcattgcgca attctcgatc agttgcgtaa tctatgctgc attgcgcaat 3960

tcttgatcag ttgcgtaatc tatgctgcat tgcgcaattc tctatatata cacatatata 4020

taaaatatat atacacacat atatataaaa tatatatata cacacatata tataaaatat 4080

atatatacac atatatataa aatatatata tacacatata tataaaatat atatatatac 4140

acatatatat aaaatatata tacacacata tatataaagt atatatatac acacatatat 4200

ataaaatata tatatacaca tatatataaa atatatatat acacatatat ataaaatata 4260

tatatacaca tatatataaa aatatatata tatatttttt aaaatattcc aattgtctca 4320

ctttgtggat gagaaaaaga agtagttaga ggtcaagtaa cttggcctac atcttttctc 4380

aagattgtaa actcctagtg agcaataacc acatcttcat tttctttgta taaaacaaga 4440

aagtttagca tgaaaaaggt actcaattac aaatgtgttg gattgaattg aagacccttg 4500

gaaggggatt ttgtacctga ggatctcttt cttttggcca tattgttcaa tggacaaaat 4560

ttagccttcg aaggcaggcc gatttgaggt taatactacc tttaccactt gatagctatg 4620

tgaccttggc catgtggttt caacagtctg aacctcattt tctctgtgta tgtgtggtcc 4680

tccttacaag tttgtgaaaa atgtgaagtc cttagccatg atagcccaat ataacaggct 4740

aaatgataat aggtttatgt tcttttcctt tatattctca gataagcact gtccgatcag 4800

ttgcgtaatc tatgctgcat tgcgcaattc tagatcagtt gcgtaatcta tgctgcattg 4860

cgcaattctt gatcagttgc gtaatctatg ctgcattgcg caattctcga tcagttgcgt 4920

aatctatgct gcattgcgca attcttgatc agttgcgtaa tctatgctgc attgcgcaat 4980

tctctcttta attctaatat atttagaatt catctttaat tctaatatat ttagaattct 5040

tctttaattc taatatattt agaattcctc tttaattcta atatatttag aattcttctt 5100

taattctaat atatttagaa ttccgctagc tgaggctccg gtgcccgtca gtgggcagag 5160

cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc 5220

tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt 5280

cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc 5340

aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc 5400

tttacgggtt atggcccttg cgtgccttga attacttcca cctggctcca gtacgtgatt 5460

cttgatcccg agctggagcc aggggcgggc cttgcgcttt aggagcccct tcgcctcgtg 5520

cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc 5580

gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg 5640

cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaggatctg cacactggta 5700

tttcggtttt tgggcccgcg gccggcgacg gggcccgtgc gtcccagcgc acatgttcgg 5760

cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc 5820

ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc 5880

tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctccag 5940

ggggctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa 6000

ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc 6060

cgtccaggca cctcgattag ttctggagct tttggagtac gtcgtcttta ggttgggggg 6120

aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag 6180

cttggcactt gatgtaattc tcgttggaat ttgccctttt tgagtttgga tcttggttca 6240

ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaacac 6300

gtggtcgcgg ccgcttcgaa ggtaccatgg agaaaaaaat cactggatat accaccgttg 6360

atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta 6420

cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata 6480

agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg 6540

aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt 6600

acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg 6660

atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg 6720

cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga 6780

gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca 6840

ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc 6900

atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact 6960

gcgatgagtg gcagggcggg gcgtaaagat ctggccggct gggcccgttt cgaaggtaag 7020

cctatcccta accctctcct cggtctcgat tctacgcgta ccggtcatca tcaccatcac 7080

cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 7140

gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 7200

taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 7260

ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat 7320

gcggtgggct ctatggatat cgcttctgag gcggaaagaa ccagtggcgg taatacggtt 7380

atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 7440

caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 7500

gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 7560

ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 7620

cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 7680

taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 7740

cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 7800

acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 7860

aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 7920

atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 7980

atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 8040

gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 8100

gtggaacgaa aactcacgtt aagggatttt ggtcatgaca ttaacctata aaaataggcg 8160

tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 8220

gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 8280

tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga 8340

gcagattgta ctgagagtgc accatatatg cggtgtgaaa taccgcacag atgcgtaagg 8400

agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcatctttaa ttctaatata 8460

tttagaattc atctttaatt ctaatatatt tagaattctt ctttaattct aatatattta 8520

gaattcctct ttaattctaa tatatttaga attcttcttt aattctaata tatttagaat 8580

tccgatcagt tgcgtaatct atgctgcatt gcgcaattct agatcagttg cgtaatctat 8640

gctgcattgc gcaattcttg atcagttgcg taatctatgc tgcattgcgc aattctcgat 8700

cagttgcgta atctatgctg cattgcgcaa ttcttgatca gttgcgtaat ctatgctgca 8760

ttgcgcaatt ctctatatat acacatatat ataaaatata tatacacaca tatatataaa 8820

atatatatat acacacatat atataaaata tatatataca catatatata aaatatatat 8880

atacacatat atataaaata tatatatata cacatatata taaaatatat atacacacat 8940

atatataaag tatatatata cacacatata tataaaatat atatatacac atatatataa 9000

aatatatata tacacatata tataaaatat atatatacac atatatataa aaatatatat 9060

atatattttt taaaatattc caattgtctc actttgtgga tgagaaaaag aagtagttag 9120

aggtcaagta acttggccta catcttttct caagattgta aactcctagt gagcaataac 9180

cacatcttca ttttctttgt ataaaacaag aaagtttagc atgaaaaagg tactcaatta 9240

caaatgtgtt ggattgaatt gaagaccctt ggaaggggat tttgtacctg aggatctctt 9300

tcttttggcc atattgttca atggacaaaa tttagccttc gaaggcaggc cgatttgagg 9360

ttaatactac ctttaccact tgatagctat gtgaccttgg ccatgtggtt tcaacagtct 9420

gaacctcatt ttctctgtgt atgtgtggtc ctccttacaa gtttgtgaaa aatgtgaagt 9480

ccttagccat gatagcccaa tataacaggc taaatgataa taggtttatg ttcttttcct 9540

ttatattctc agataagcac tgtccgatca gttgcgtaat ctatgctgca ttgcgcaatt 9600

ctagatcagt tgcgtaatct atgctgcatt gcgcaattct tgatcagttg cgtaatctat 9660

gctgcattgc gcaattctcg atcagttgcg taatctatgc tgcattgcgc aattcttgat 9720

cagttgcgta atctatgctg cattgcgcaa ttctctcttt aattctaata tatttagaat 9780

tcatctttaa ttctaatata tttagaattc ttctttaatt ctaatatatt tagaattcct 9840

ctttaattct aatatattta gaattcttct ttaattctaa tatatttaga attcctgcgc 9900

aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgcca 9947

<210>7

<211>7031

<212>DNA

<213>Synthesized DNA

<400>7

gcgcgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 60

ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 120

gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 180

caatagggac tttccattga cgtcaatggg tggactattt acggtaaact gcccacttgg 240

cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 300

ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 360

tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 420

gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 480

gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 540

tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 600

taactagaga acccactgcttactggctta tcgaaattaa tacgactcac tatagggaga 660

cccaagcttg cattcctgca ggtcgacatc gatcttaagc agtacttcta gaggatccga 720

acaaaaactc atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca 780

ttgagtttga tccccgggaa ttcagacatg ataagataca ttgatgagtt tggacaaacc 840

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 900

tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag 960

atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct 1020

ttcatagaag gcggcggtgg aatcgaaatc tcgtagcacg tgtagcgctt tagaaaaact 1080

catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 1140

gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 1200

gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 1260

cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 1320

agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct 1380

cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 1440

gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 1500

gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 1560

cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 1620

ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 1680

tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 1740

catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 1800

cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgagcaag 1860

acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca 1920

gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag 1980

acacaacgtg gctttccccc cccccccatt attgaagcat ttatcagggt tattgtctca 2040

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 2100

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 2160

aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 2220

tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 2280

gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 2340

cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat 2400

gcgtaaggag aaaataccgc atcagattgg ctattggcca ttgcatacgt tgtatccata 2460

tcataatatg tacaattcgc ccttcattta attctaccgg gtaggggagg cgcttttccc 2520

aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 2580

cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 2640

tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 2700

gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 2760

ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 2820

ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 2880

gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 2940

gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttctcg 3000

agctcaagct tggggggggg gacagctcag ggctgcgatt tcgcgccaaa cttgacggca 3060

atcctagcgt gaaggctggt aggattttat ccccgctgcc atcatggttc gaccattgac 3120

tgcatcgtcg ccgtgtccca aaatatgggg attggcaaga acggagacct accctggcct 3180

ccgctcagga acgagttcaa gtacttccaa agaatgacca caacctcttc agtggaaggt 3240

aaacagaatc tggtgattat gggtaggaaa acctggttct ccattcctga gaagaatcga 3300

cctttaaagg acagaattaa tatagttctc agtagagaac tcaaagaacc accacgagga 3360

gctcattttc ttgccaaaag tttggatgat gccttaagac ttattgaaca accggaattg 3420

gcaagtaaag tagacatggt ttggatagtc ggaggcagtt ctgtttacca ggaagccatg 3480

aatcaaccag gccacctcag actctttgtg acaaggatca tgcaggaatt tgaaagtgac 3540

acgtttttcc cagaaattga tttggggaaa tataaacttc tcccagaata cccaggcgtc 3600

ctctctgagg tccaggagga aaaaggcatc aagtataagt ttgaagtcta cgagaagaaa 3660

gactaagcta gctgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc 3720

cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg 3780

gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa 3840

ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga 3900

acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct 3960

tgcgtgcctt gaattacttc cacctggctc cagtacgtga ttcttgatcc cgagctggag 4020

ccaggggcgg gccttgcgct ttaggagccc cttcgcctcg tgcttgagtt gaggcctggc 4080

ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 4140

tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 4200

aagatagtct tgtaaatgcg ggccaggatc tgcacactgg tatttcggtt tttgggcccg 4260

cggccggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 4320

cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 4380

gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 4440

ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctcc agggggctca aaatggagga 4500

cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 4560

cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 4620

agttctggag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 4680

agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 4740

tctcgttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 4800

tggttcaaag tttttttctt ccatttcagg tgtcgtgaac acgtggtcgc ggccgcttcg 4860

aaggtaccat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc 4920

gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc 4980

agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg 5040

cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga 5100

aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc 5160

aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac 5220

acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt 5280

ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt 5340

taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata 5400

cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg 5460

gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg 5520

gggcgtaaag atctggccgg ctgggcccgt ttcgaaggta agcctatccc taaccctctc 5580

ctcggtctcg attctacgcg taccggtcat catcaccatc accattgagt ttaaacccgc 5640

tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 5700

ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 5760

gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 5820

aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggat 5880

atcgcttctg aggcggaaag aaccagtggc ggtaatacgg ttatccacag aatcagggga 5940

taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 6000

cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 6060

ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 6120

aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 6180

tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 6240

gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 6300

cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 6360

ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 6420

cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 6480

gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 6540

cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 6600

tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 6660

ttaagggatt ttggtcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 6720

tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 6780

cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 6840

tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt 6900

gcaccatata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg 6960

cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 7020

ctattacgcc a 7031

Claims (12)

1. An isolated MAR core fragment of a recombinant expression vector, having a nucleotide base sequence as set forth in SEQ ID NO: 2, respectively.

2. A recombinant expression vector comprising the MAR core fragment of claim 1.

3. The vector of claim 2, wherein said MAR core fragment is inserted upstream of a promoter and downstream of a polyadenylation site.

4. The vector of claim 3, wherein the promoter is CMV, SV40 or EF-1a promoter.

5. The vector of any one of claims 2-4, which is pBudCE4.1, pDR1, pcDNA3.1(+), pDFHF or pCHO1.0, or a modified vector of any of the foregoing vectors.

6. The vector of claim 5, which is a pBudCE4.1 modified vector, and the nucleotide sequence of the vector is shown in SEQ ID NO: 5, respectively.

7. The vector of any one of claims 2-4, wherein the vector comprises one or more genes encoding one or more recombinant proteins.

8. The vector of claim 5, wherein the vector comprises one or more genes encoding one or more recombinant proteins.

9. The vector of claim 6, wherein the vector comprises one or more genes encoding one or more recombinant proteins.

10. A host cell comprising the vector of any one of claims 7-9.

11. The cell of claim 10, which is a mammalian cell selected from the group consisting of: chinese Hamster Ovary (CHO) cells, COS cells, Vero cells, SP2/0 cells, NS/0 myeloma cells, human embryonic kidney (HEK 293) cells, Baby Hamster Kidney (BHK) cells, Hela cells, human B cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells or PerC6 cells and MDCK cells.

12. A method for producing a recombinant protein, said method comprising transcribing and translating one or more genes encoding said recombinant protein present in the expression vector of any one of claims 7-9.

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