CN109576269A - A kind of animal cell expression vectors of the novel core fragment containing MAR - Google Patents

Abstract

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

Description

A kind of animal cell expression vectors of the novel core fragment containing MAR

The application is that " a kind of novel contains for No. 201410129979.2 applications for a patent for invention submitting on April 1st, 2014 The divisional application of the animal cell expression vectors of MAR core fragment ".

Technical field

The present invention relates to field of biotechnology, more specifically, the animal for disclosing a kind of novel core fragment containing MAR is thin Cellular expression carrier.

Background technique

Currently, many carriers for producing recombinant protein have been developed that, recombinant protein with bacterium, eukaryotic microorganisms, Insect cell etc. is can obtain higher yield in the expression of host, but these expression systems lack similar to mammalian cell Protein modification mechanism (such as glycosylation modified etc.), the animal protein of expression easily lack bioactivity or because fold mistake due to Generate inclusion body.And recombinant protein is produced with mammalian cell expression system, yield is often lower, and quiding gene Stability also have certain problem.Later it has been found that Chinese hamster ovary cell (CHO) is production monoclonal antibody etc. The optimizer system of complicated macromolecular produces monoclonal antibody using CHO expression system, and yield is greatly improved, still Screening and to maintain overexpression cell line be still that part takes time and effort and the uncertain work of result.After cell transfecting, fusion is logical Normal random integration enters in the genome of host cell, and most of recombination betides " quiet zone ", such as nontranscribed domain.Usually need The fine cell strain of several yield can just be obtained by screening thousands of clones.

Matrix attachment region (matrix attachment region, abbreviation MAR) element be it is a kind of can help generate simultaneously Maintain the DNA sequence dna of open Chromodomain.Open Chromodomain can promote to transcribe, and the multicopy of expression and transgenosis is whole It closes.MAR element is put into ratio (Kwaks TH, the Otte AP of the yield and high yielding cell sarain that can increase cell strain in carrier 2006Trends Biotechnol 24:137-142), but MAR element is larger (3kb or more), and the efficiency of plasmid-transfected cells (Yin W et al Anal Biochem.2005Nov15 is reduced with the increase of plasmid size；346 (2): 289-94), from And limit application of the MAR element in Bicistronic expression vector.

Therefore, a kind of more efficiently mammalian cell expression vector is developed, is always those skilled in the art's urgency Problem to be solved.

Summary of the invention

The object of the present invention is to provide a kind of expression vector that quiding gene can be improved and express in mammalian cells, Contain the MAR core fragment of the matrix attachment region of manual transcription factor binding site in the expression vector both ends.The expression carries Body can effectively improve albumen yield in mammalian cells and reduce production cost.

Specifically, disclosure of the invention:

1, a kind of MAR core fragment of isolated recombinant expression carrier, nucleotide base sequence such as SEQ ID NO:1, Shown in SEQ ID NO:2 or SEQ ID NO:3.

2, a kind of recombinant expression carrier, the carrier both ends contain above-mentioned 1 described in MAR core fragment.

3, above-mentioned 2 carrier, the MAR core fragment are inserted in upstream and the site of polyadenylation of promoter Downstream.

4, above-mentioned 3 carrier, the promoter are CMV, SV40 or EF-1a promoter.Preferably, the MAR core Segment is inserted in the carrier upstream site FSPI, or the downstream SV40PA Afe I site, or the upstream PEF-1a Nhe I site, or BGH PA The downstream site EcoRV.The most preferably site upstream FSPI and the upstream PEF-1a Nhe I site.

5, any carrier of above-mentioned 2-4, the carrier be pBudCE4.1, pDR1, pcDNA3.1 (+), pDHFF or The transformation carrier of pCHO 1.0 or above-mentioned carrier.

6, above-mentioned 5 carrier, the carrier are the carrier of pBudCE4.1 transformation, the vector nucleotide sequence such as SEQ Shown in ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

7, any carrier of above-mentioned 2-6, wherein the carrier includes one or more one or more recombination eggs of coding White function integrity gene.

8, a kind of host cell, it includes carriers described in above-mentioned 7.

9, cell described in above-mentioned 8 is mammalian cell, is including but not limited to selected from group as described below: China Hamster Qvary (CHO) cell, COS cell, Vero cell, SP2/0 cell, NS/0 myeloma cell, Human embryo kidney (HEK 293) Cell, baby hamster kidney (BHK) cell, Hela cell, human B cell, CV-1/EBNA cell, L cell, 3T3 cell, HEPG2 are thin Born of the same parents, PerC6 cell and mdck cell.

10, a kind of method of preparation and reorganization albumen, the method includes transcription and translations to be present in any of the above-described table The gene of the recombinant protein is encoded up to the one or more on carrier.

Expression vector of the invention is particularly suitable for application to mammalian cell, especially Chinese hamster ovary cell CHO Expression.

Both ends of the invention can be inserted into lactation added with the MAR core fragment DNA sequence dna of manual transcription factor binding site and move Object fibrocyte expression vector, so that the mammalian genes for greatly improving importing are expressed in mammalian cells, and stability is very It is good.

The present invention provides a kind of mammalian cell expression vector, the expression vector contains both ends added with manual transcription For the MAR core fragment DNA sequence dna of factor binding site as present example is enumerated, the expression vector is transformation PBudCE4.1 carrier, the pBudCE4.1 carrier of the transformation, in SV40 site of polyadenylation and BGH site of polyadenylation Afe I and EcoR V restriction site is inserted into downstream respectively.With kanamycin resistant gene and DHFR gene replacement Zeocin resistant gene in pBudCE4.1.The transformation carrier is named as pBCM1.1, pBCM1.2, pBCM1.3, DNA sequence Column are respectively SEQ ID No.4, shown in 5,6.Without above-mentioned matrix attachment region element segment and manual transcription factor bound site The transformation carrier of point DNA is named as pBCM1.0, and DNA sequence dna is as shown in SEQ ID No 7.

Both ends of the invention can use conventional conjunction added with the MAR core fragment DNA sequence dna of manual transcription factor binding site It is prepared at method.

Plasmid by both ends of the invention added with the MAR core fragment of manual transcription factor binding site adds with without both ends There is the plasmid of the MAR core fragment of artificial Binding site for transcription factor to compare.By measurement foreign protein in above two matter The expression quantity of grain, the results showed that, both ends of the invention are special added with the plasmid of the MAR core fragment of manual transcription factor binding site Not Shi Yongyu Chinese hamster ovary celI, with without both ends added with manual transcription factor binding site matrix attachment region element MAR core sheet The plasmid of section is compared, and the expression quantity of the foreign protein in zooblast is greatly improved, and expression quantity improves 10-17 times.It is high The ratio for producing cell strain improves 25-40 times.

The gene of foreign protein (such as human cytokines, antibody) is inserted into expression vector of the invention, is then transfected To mammalian cell, it can be used for the production of foreign protein (such as human cytokines, antibody).

Detailed description of the invention

Expression vector of the attached drawing 1 containing MAR core fragment of the present invention with without MAR gene or the full-length gene order containing MAR Expression vector is averaged GFP expression quantity comparison diagram.

Expression vector of the attached drawing 2 containing MAR core fragment of the present invention with without MAR gene or the full-length gene order containing MAR The GFP of expression vector is averaged overexpression cell line ratio comparison diagram.

Expression vector of the attached drawing 3 containing MAR core fragment of the present invention with without MAR gene or the full-length gene order containing MAR Expression vector is averaged CAT expression quantity comparison diagram.

Expression vector of the attached drawing 4 containing MAR core fragment of the present invention with without MAR gene or the full-length gene order containing MAR The CAT of expression vector is averaged overexpression cell line ratio comparison diagram.

Attached drawing 5pBCM1.0 plasmid construct figure.

Specific embodiment

Following embodiment, experimental example are that the present invention is further detailed, and be should not be construed as limiting the invention.

Expression vector cited by the embodiment of the present invention (is purchased from: Life by pBudCE4.1 carrier Technologies) transformation obtains: being included in SV40 site of polyadenylation and BGH site of polyadenylation downstream is inserted into respectively AfeI and EcoR V restriction site；By kalamycin resistance gene (Kanamycin Resistance Gene), PGK is opened Mover, dihyrofolate reductase (DHFR) and the SV40PA PCR from pCHO1.0 (being purchased from Life Technologies) go out, and It is fitted into pBudCE4.1 by the site NheI and AfeI, and removes Zeocin resistant gene simultaneously；By chloramphenicol acetyltransferase Gene (Chloramphenicol Acetyltransferase gene, CAT gene) (is purchased from from pBudCE4.1/lacZ/CAT Life Technologies) in PCR go out, and be fitted into pBudCE4.1 by the site Kpn I and Bgl II；By green fluorescence egg White gene (Green Fluorescent Protein gene, GFP gene) (is purchased from Life from PCDNA3.1/CT-GFP-TOP9 Technologies PCR goes out in), and is packed into pBudCE4.1 by Pst I and BamH I site.

Afe I and EcoR V restriction site are introduced in embodiment 1pBudCE4.1

By the method for point mutation by Afe I and EcoR V restriction site introduce SV40 site of polyadenylation and BGH site of polyadenylation downstream.A little dash forward with Quickchange point mutation kit (being purchased from Agilent Technologies) Become.Introduce Afe I restriction enzyme site.

Forward primer: 5 '-GGAAAACGATTCCGAAGC GCT AC-3 '

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

Rite-directed mutagenesis PCR amplification condition is as follows: 1,95 DEG C 30 seconds；2,95 DEG C 30 seconds；3,55 DEG C 1 minute；4,68℃ 5 Minute；5,2-4 repeats 18 circulations.

Dpn I enzyme is added to digest template plasmid, then converts XL1-Blue competent E.coli.Specific steps are such as Under: 1. it is freshly prepared or -20 DEG C at the 100uL competent cell (purchased from Agilent Technologies) that saves, be placed on ice, solve completely Lightly by cell even suspension after ice.2. Dpn I enzyme (being purchased from: NEB company) postdigestive plasmid is added, mix gently.3. It places 30 minutes on ice.4.42 DEG C water-bath heat shock 60 seconds.5. placing 2 minutes on ice.6. 400uL LB culture solution is added (to be purchased from Sigma-Aldrich), 37 DEG C 250 revs/min of shaken cultivations 30 minutes.7, that bacterium is coated on Amp/LB agar plate 8. is flat Ware is positive at 37 DEG C to be placed 1 hour, and plate is inverted, overnight incubation by liquid absorption to be seeded into after agar.9, through 37 DEG C Overnight incubation, the bacterium colony that occurs on ampicillin/LB agar plate be plasmid conversion Escherichia coli, 10, picking Monoclonal bacterium simultaneously extracts plasmid.After the plasmid being mutated, identify correctly and serve Hai Shenggong bioengineering have with Afe I digestion The sequencing of limit company, as a result correctly.

Then EcoR V restriction enzyme site is introduced in the carrier for having been introduced into AfeI:

Forward primer: 5 '-ATCCACAGAATCAGGGGATAT CGC-3 '

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

Rite-directed mutagenesis PCR amplification condition is as follows: 1,95 DEG C 30 seconds.2,95 DEG C 30 seconds；3,55 DEG C 1 minute；4,68℃ 5 Minute；5,2-4 repeats 18 circulations

Dpn I enzyme is added to digest template plasmid, then converts XL1-Blue competent E.coli.The matter being mutated It identifies correct with EcoR V digestion after grain and serves the sequencing of Hai Shenggong bioengineering Co., Ltd, as a result correctly.

Zeocin resistant gene is removed in embodiment 2pBudCE4.1 and kalamycin resistance gene is added, PGK promoter, DHFR and SV40PA

Kalamycin resistance gene, PGK promoter, DHFR and SV40PA (are purchased from Life from pCHO1.0 Technologies PCR goes out in), and is packed into the pBudCE4.1 obtained of embodiment 1 by the site NheI and AfeI, goes simultaneously Except Zeocin resistant gene.

Forward primer: 5 '-GTG AGCGCTTTAGAAAAACTCATCGAGCATC-3 '

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

PCR with Primestar HS high-fidelity DNA polymerase (be purchased from TAKARA company),

Specific step is as follows: 1,95 DEG C 3 minutes；2,98 DEG C 10 seconds；3,47 DEG C 15 seconds；4,72 DEG C 3 minutes；5,2-4 Repeat 30 circulations；6,72 DEG C 5 minutes.

Improved new support is named as PBCM1.0, and structure chart is shown in attached drawing 5, wherein NheI, AfeI, FspI and The site EcoRV is insertion point of the both ends added with the MAR core fragment of manual transcription factor binding site.

The building of embodiment 3 is containing both ends added with the expression vector two of the MAR core fragment of manual transcription factor binding site Hold MAR core fragment (the sequence such as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID added with manual transcription factor binding site Shown in NO:3) it synthesizes to obtain by Shanghai Sheng Gong bioengineering Co., Ltd.

Both ends can be inserted in the upstream the PBCM1.0 site FSPI added with the MAR core fragment of manual transcription factor binding site, or The downstream SV40PA Afe I site, or the upstream PEF-1a Nhe I site, or the downstream the BGH PA site EcoRV.

In the following examples, both ends are inserted in PBCM 1.0 added with the MAR core fragment of manual transcription factor binding site The upstream site FSPI and the upstream PEF-1a Nhe I site, MAR core fragment sequence such as SEQ ID NO:1, SEQ ID NO:2 or Shown in SEQ ID NO:3.

MAR core fragment by both ends added with manual transcription factor binding site is connected to the upstream PBCM1.0 by flush end The site FSPI.And it is sequenced correct.

MAR core fragment by both ends added with manual transcription factor binding site is connected on PBCM1.0 by viscous end Swim Nhe I site.And it is sequenced correct.

Obtained both ends are constructed to name respectively added with the expression vector of the MAR core fragment of manual transcription factor binding site For pBCM1.1, pBCM1.2, pBCM1.3, DNA sequence dna is respectively SEQ ID No.4, and 5,6.

Embodiment 4 in expression vector of the both ends added with the MAR core fragment of manual transcription factor binding site containing being inserted into Foreign protein genes

The site KpnI and BglII in the CAT gene insertion downstream PEF-1a

By CAT gene from pBudCE4.1/lacZ/CAT (Life Technologies) PCR go out and pass through Kpn I and The site Bgl II is packed into expression vector constructed by above-described embodiment 3.

Forward primer: 5 '-GTG GGTACC ATGGAGAAAAAAATCACTGG-3 '

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

PCR with Primestar HS high-fidelity DNA polymerase (be purchased from TAKARA company),

Specific step is as follows: 95 DEG C 3 minutes；2,98 DEG C 10 seconds；3,52 DEG C 15 seconds；4,72 DEG C 1 minute, 2-4 are repeated 30 circulations；5,72 DEG C 5 minutes

The expression quantity of CAT gene is with chloramphenicol acetyltransferase detection kit (be purchased from Life Technologies) Measurement.

GFP gene PCR from PCDNA3.1/CT-GFP-TOP9 (being purchased from Life Technologies) goes out and passes through downstream Pst I, BamH I site is packed into expression vector constructed by embodiment 3.

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

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

PCR with Primestar HS high-fidelity DNA polymerase (be purchased from TAKARA company),

Specific step is as follows: 95 DEG C 3 minutes；2,98 DEG C 10 seconds；3,50 DEG C 15 seconds；4,72 DEG C 1 minute, 2-4 are repeated 30 circulations；5,72 DEG C 5 minutes

The expression quantity of GFP can pass through cytofluorometric analyses (BD FACSCalibur flow Cytometer) (BD company is purchased from) to measure and analyze by 2.8 software of WinMDI.

Embodiment 5 containing both ends added with the MAR core fragment of manual transcription factor binding site carrier express GFP and CAT

MAR core fragment (sequence such as SEQ ID NO:1, SEQ ID of the both ends added with manual transcription factor binding site Shown in NO:2 or SEQ ID NO:3) to increase Green Fluorescent Protein and the chloramphenicol acetyltransferase table in the Chinese hamster ovary celI of transfection Up to horizontal influence.

The table that will be built in embodiment 4 using FreeStyle MAX transfection reagent (being purchased from Life Technologies) It is imported in CHO-S cell (being purchased from Life Technologies) up to carrier pBCM1.1, pBCM1.2, pBCM1.3.By above-mentioned turn Cell sets 37 DEG C after dye, 8%CO₂, 130-150rpm, which suspends, to be cultivated.Start to stablize expression screening after 48 hours.Simultaneously to be free of The expression vector of MAR and the full length sequence containing MAR is as control.Experimental result is as shown in attached drawing 1-4, the results showed that, containing the present invention The expression quantity of foreign protein of the expression vector in zooblast of MAR core fragment greatly improved, GFP expression quantity mentions High 10-17 times (see attached drawing 1).CAT expression quantity improves 14-15 times (see attached drawing 3).The ratio of GFP high yielding cell sarain improves 25-40 times (see attached drawing 2).The ratio of CAT high yielding cell sarain improves 25-40 times (see attached drawing 4).

Sequence table

<110>three lives state is good for medicine company (Shanghai) limited liability company

<120>a kind of animal cell expression vectors of the novel core fragment containing MAR

<130> 1

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1314

<212> DNA

<213> Synthesized DNA

<400> 1

gtggctagct ctttaattct aatatattta gaattcatct ttaattctaa tatatttaga 60

attcttcttt aattctaata tatttagaat tcctctttaa ttctaatata tttagaattc 120

ttctttaatt ctaatatatt tagaattcct ctttaattct aatatattta gaattcatct 180

ttaattctaa tatatttaga attcctcttt aattctaata tatttagaat tcttctttaa 240

ttctaatata tttagaattc atatatatac acatatatat aaaatatata tacacacata 300

tatataaaat atatatatac acacatatat ataaaatata tatatacaca tatatataaa 360

atatatatat acacatatat ataaaatata tatatataca catatatata aaatatatat 420

acacacatat atataaagta tatatataca cacatatata taaaatatat atatacacat 480

atatataaaa tatatatata cacatatata taaaatatat atatacacat atatataaaa 540

atatatatat atatttttta aaatattcca attgtctcac tttgtggatg agaaaaagaa 600

gtagttagag gtcaagtaac ttggcctaca tcttttctca agattgtaaa ctcctagtga 660

gcaataacca catcttcatt ttctttgtat aaaacaagaa agtttagcat gaaaaaggta 720

ctcaattaca aatgtgttgg attgaattga agacccttgg aaggggattt tgtacctgag 780

gatctctttc ttttggccat attgttcaat ggacaaaatt tagccttcga aggcaggccg 840

atttgaggtt aatactacct ttaccacttg atagctatgt gaccttggcc atgtggtttc 900

aacagtctga acctcatttt ctctgtgtat gtgtggtcct ccttacaagt ttgtgaaaaa 960

tgtgaagtcc ttagccatga tagcccaata taacaggcta aatgataata ggtttatgtt 1020

cttttccttt atattctcag ataagcactg tcctctttaa ttctaatata tttagaattc 1080

atctttaatt ctaatatatt tagaattctt ctttaattct aatatattta gaattcctct 1140

ttaattctaa tatatttaga attcttcttt aattctaata tatttagaat tcctctttaa 1200

ttctaatata tttagaattc atctttaatt ctaatatatt tagaattcct ctttaattct 1260

aatatattta gaattcttct ttaattctaa tatatttaga attcagctag cgtg 1314

<210> 2

<211> 1494

<212> DNA

<213> Synthesized DNA

<400> 2

gtggctagcg atcagttgcg taatctatgc tgcattgcgc aattctagat cagttgcgta 60

atctatgctg cattgcgcaa ttcttgatca gttgcgtaat ctatgctgca ttgcgcaatt 120

ctcgatcagt tgcgtaatct atgctgcatt gcgcaattct tgatcagttg cgtaatctat 180

gctgcattgc gcaattctcg atcagttgcg taatctatgc tgcattgcgc aattctagat 240

cagttgcgta atctatgctg cattgcgcaa ttctcgatca gttgcgtaat ctatgctgca 300

ttgcgcaatt cttgatcagt tgcgtaatct atgctgcatt gcgcaattct atatatatac 360

acatatatat aaaatatata tacacacata tatataaaat atatatatac acacatatat 420

ataaaatata tatatacaca tatatataaa atatatatat acacatatat ataaaatata 480

tatatataca catatatata aaatatatat acacacatat atataaagta tatatataca 540

cacatatata taaaatatat atatacacat atatataaaa tatatatata cacatatata 600

taaaatatat atatacacat atatataaaa atatatatat atatttttta aaatattcca 660

attgtctcac tttgtggatg agaaaaagaa gtagttagag gtcaagtaac ttggcctaca 720

tcttttctca agattgtaaa ctcctagtga gcaataacca catcttcatt ttctttgtat 780

aaaacaagaa agtttagcat gaaaaaggta ctcaattaca aatgtgttgg attgaattga 840

agacccttgg aaggggattt tgtacctgag gatctctttc ttttggccat attgttcaat 900

ggacaaaatt tagccttcga aggcaggccg atttgaggtt aatactacct ttaccacttg 960

atagctatgt gaccttggcc atgtggtttc aacagtctga acctcatttt ctctgtgtat 1020

gtgtggtcct ccttacaagt ttgtgaaaaa tgtgaagtcc ttagccatga tagcccaata 1080

taacaggcta aatgataata ggtttatgtt cttttccttt atattctcag ataagcactg 1140

tccgatcagt tgcgtaatct atgctgcatt gcgcaattct agatcagttg cgtaatctat 1200

gctgcattgc gcaattcttg atcagttgcg taatctatgc tgcattgcgc aattctcgat 1260

cagttgcgta atctatgctg cattgcgcaa ttcttgatca gttgcgtaat ctatgctgca 1320

ttgcgcaatt ctcgatcagt tgcgtaatct atgctgcatt gcgcaattct agatcagttg 1380

cgtaatctat gctgcattgc gcaattctcg atcagttgcg taatctatgc tgcattgcgc 1440

aattcttgat cagttgcgta atctatgctg cattgcgcaa ttctagctag cgtg 1494

<210> 3

<211> 1470

<212> DNA

<213> Synthesized DNA

<400> 3

gtggctagct ctttaattct aatatattta gaattcatct ttaattctaa tatatttaga 60

attcttcttt aattctaata tatttagaat tcctctttaa ttctaatata tttagaattc 120

ttctttaatt ctaatatatt tagaattccg atcagttgcg taatctatgc tgcattgcgc 180

aattctagat cagttgcgta atctatgctg cattgcgcaa ttcttgatca gttgcgtaat 240

ctatgctgca ttgcgcaatt ctcgatcagt tgcgtaatct atgctgcatt gcgcaattct 300

tgatcagttg cgtaatctat gctgcattgc gcaattctct atatatacac atatatataa 360

aatatatata cacacatata tataaaatat atatatacac acatatatat aaaatatata 420

tatacacata tatataaaat atatatatac acatatatat aaaatatata tatatacaca 480

tatatataaa atatatatac acacatatat ataaagtata tatatacaca catatatata 540

aaatatatat atacacatat atataaaata tatatataca catatatata aaatatatat 600

atacacatat atataaaaat atatatatat attttttaaa atattccaat tgtctcactt 660

tgtggatgag aaaaagaagt agttagaggt caagtaactt ggcctacatc ttttctcaag 720

attgtaaact cctagtgagc aataaccaca tcttcatttt ctttgtataa aacaagaaag 780

tttagcatga aaaaggtact caattacaaa tgtgttggat tgaattgaag acccttggaa 840

ggggattttg tacctgagga tctctttctt ttggccatat tgttcaatgg acaaaattta 900

gccttcgaag gcaggccgat ttgaggttaa tactaccttt accacttgat agctatgtga 960

ccttggccat gtggtttcaa cagtctgaac ctcattttct ctgtgtatgt gtggtcctcc 1020

ttacaagttt gtgaaaaatg tgaagtcctt agccatgata gcccaatata acaggctaaa 1080

tgataatagg tttatgttct tttcctttat attctcagat aagcactgtc cgatcagttg 1140

cgtaatctat gctgcattgc gcaattctag atcagttgcg taatctatgc tgcattgcgc 1200

aattcttgat cagttgcgta atctatgctg cattgcgcaa ttctcgatca gttgcgtaat 1260

ctatgctgca ttgcgcaatt cttgatcagt tgcgtaatct atgctgcatt gcgcaattct 1320

ctctttaatt ctaatatatt tagaattcat ctttaattct aatatattta gaattcttct 1380

ttaattctaa tatatttaga attcctcttt aattctaata tatttagaat tcttctttaa 1440

ttctaatata tttagaattc cgctagcgtg 1470

<210> 4

<211> 9635

<212> DNA

<213> Synthesized DNA

<400> 4

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 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 gcggtggttt 7860

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 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 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 (10)

1. a kind of MAR core fragment of isolated recombinant expression carrier, nucleotide base sequence is as shown in SEQ ID NO:2.

2. a kind of recombinant expression carrier, the carrier is containing MAR core fragment described in claim 1.

3. carrier described in claim 2, the upstream of the MAR core fragment MAR core fragment insertion promoter and poly- adenosine The downstream of polyadenylation sites.

4. carrier described in claim 3, the promoter is CMV, SV40 or EF-1a promoter.

5. any carrier of claim 2-4, the carrier is pBudCE4.1, pDR1, pcDNA3.1 (+), pDHFF or The transformation carrier of pCHO 1.0 or any of the above-described carrier.

6. carrier described in claim 5, for the carrier of pBudCE4.1 transformation, the vector nucleotide sequence such as SEQ ID Shown in NO:5.

7. any carrier of claim 2-6, wherein the carrier includes one or more one or more recombination eggs of coding White gene.

8. a kind of host cell, it includes carriers as claimed in claim 7.

9. cell according to any one of claims 8 is mammalian cell, can be selected from: Chinese hamster ovary (CHO) cell, COS are thin Born of the same parents, Vero cell, SP2/0 cell, NS/0 myeloma cell, Human embryo kidney (HEK 293) cell, baby hamster kidney (BHK) cell, Hela cell, human B cell, CV-1/EBNA cell, L cell, 3T3 cell, HEPG2 cell or PerC6 cell and MDCK are thin Born of the same parents.

10. a kind of method of preparation and reorganization albumen is present in any of the above-described expression the method includes transcription and translation and carries One or more on body encodes the gene of the recombinant protein.