质粒名称: | pCW57-MCS1-P2A-MCS2 (Neo) |
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出品公司: | 爱迪基因 |
目录编号: | 89180 |
存储实验室: | Adam Karpf |
载体骨架基本信息 |
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载体名称: | pCW57.1 |
载体出品公司: | Broad Insitute |
载体抗性: | 氨苄青霉素 |
空载体大小: | 7943 bp |
完整质粒大小: | -- |
载体修饰: | 用rtTA-T2A-新霉素代替嘌呤霉素T2A-rtTA对pCW57-MCS1-2A-MCS2进行了改性。 |
载体类型: | 哺乳动物表达,慢病毒;强力霉素诱导;P2A自裂解肽 |
筛选标记: | 新霉素(G418) |
高拷贝/低拷贝: | -- |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | -- |
物种来源: | -- |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | TRE |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | pCW57.MCS_Seq_F CGTATGTCGAGGTAGGCGTG |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pCW57-MCS1-P2A-MCS2 (Neo) was a gift from Adam Karpf (Addgene plasmid # 89180 ; http://n2t.net/addgene:89180 ; RRID:Addgene_89180) |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pCW57-MCS1-P2A-MCS2 (Neo) 7943 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION All-in-one doxycycline inducible lentiviral vector for expression of one or two genes using the P2A self-cleaving peptide.. ACCESSION . VERSION . KEYWORDS pCW57-MCS1-P2A-MCS2 (Neo) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7943) TITLE All-in-one doxycycline inducible lentiviral vector for expression of one or two genes. JOURNAL Unpublished REFERENCE 2 (bases 1 to 7943) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7943 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 32..346 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 40..58 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 76..94 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 111..129 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 147..165 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 183..201 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 218..236 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 254..272 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 319..343 /label=LNCX /note="Human CMV promoter, forward primer" CDS 387..443 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" promoter 471..981 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 900..918 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 1005..1748 /codon_start=1 /product="improved tetracycline-controlled transactivator" /label=rtTA-Advanced /note="In the Tet-On(R) system, rtTA-Advanced binds to the Tet-responsive element and stimulates transcription only in the presence of tetracycline or doxycycline." /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA DALDDFDLDMLPADALDDFDLDMLPG" CDS 1764..1817 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 1827..2621 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1881..1900) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2491..2510 /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind complement(2662..2682) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3080..3091) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3296..3529 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3601..3722 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3638..3657) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3692..3711 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3762..3897 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3824..3843 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 3897..3968 /gene="bla" /label=AmpR promoter CDS 3969..4829 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4187..4206) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5000..5588 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5489..5508 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5742..5759 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5876..5897 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5912..5942 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5950..5966 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 5955..5977 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5974..5990 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5974..5990 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6009..6029 /label=T3 /note="T3 promoter, forward primer" promoter 6011..6029 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6057..6283 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6284..6464 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6511..6636 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7129..7362 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." misc_feature 7804..7921 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac 61 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag 121 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt 181 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt 241 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg 301 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta 361 gcgaattcgt cgacaccggt tccggagcca cgaacttctc tctgttaaag caagcaggag 421 acgtggaaga aaaccccggt cctaggctgc agacgcgttg tacaggatcc ggggttgggg 481 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt 541 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc 601 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc 661 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca 721 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg 781 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa 841 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg 901 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga 961 atcaccgacc tctctcccca gcaattcacc cgcgcgcggc caccatgtct agactggaca 1021 agagcaaagt cataaacgga gctctggaat tactcaatgg tgtcggtatc gaaggcctga 1081 cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga 1141 agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc 1201 acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat 1261 accgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa 1321 cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct 1381 ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat 1441 tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta 1501 tgcccccact tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct 1561 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg 1621 gcgggccgac cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg 1681 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc 1741 tccccgggtt cgaaggaagc ggagaaggta gaggttctct cctcacttgt ggtgatgttg 1801 aagaaaaccc tggtccaatg tctagaatga ttgaacaaga tggattgcac gcaggttctc 1861 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 1921 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 1981 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 2041 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 2101 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 2161 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 2221 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 2281 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 2341 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 2401 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 2461 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 2521 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 2581 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aatgcattga ggatcgatcc 2641 aagatatcgt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat 2701 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc 2761 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg 2821 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct 2881 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct 2941 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg 3001 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac 3061 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct 3121 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct 3181 ttgggccgcc tccccgcctg tttcgcctcg gcgtccggac tagaggtacc tttaagacca 3241 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa 3301 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg 3361 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 3421 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 3481 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca 3541 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg 3601 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 3661 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 3721 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc 3781 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 3841 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggccttt 3901 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 3961 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4021 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 4081 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 4141 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 4201 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 4261 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 4321 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 4381 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 4441 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 4501 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 4561 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 4621 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 4681 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 4741 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 4801 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 4861 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 4921 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 4981 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 5041 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 5101 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 5161 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 5221 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 5281 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 5341 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 5401 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 5461 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5521 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 5581 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 5641 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 5701 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 5761 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 5821 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 5881 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 5941 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 6001 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta 6061 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt 6121 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc 6181 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca 6241 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga 6301 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 6361 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 6421 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg 6481 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa 6541 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc 6601 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga 6661 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat 6721 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca 6781 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa 6841 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag 6901 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc 6961 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg 7021 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac 7081 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt 7141 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt 7201 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat 7261 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag 7321 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc 7381 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct 7441 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac 7501 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga 7561 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct 7621 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt 7681 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac 7741 ccacctccca accccgaggg gacaattctc gacctcgaga caaatggcag tattcatcca 7801 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 7861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt 7921 tcgggtttat tacagggaca gca //
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