质粒名称: | pSpCas9 (PX165) |
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出品公司: | Addgene |
目录编号: | 48137 |
存储实验室: | Feng Zhang |
载体骨架基本信息 |
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载体名称: | PX165 |
完整质粒大小: | 8300 bp |
载体类型: | 哺乳细胞表达,CRISPR |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | Stbl3 |
插入基因信息 |
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插入基因名称: | hSpCas9,SpCas9,Cas9,PX165 |
物种来源: | Synthetic |
插入大小: | 5400 bp |
启动子: | Cbh |
标签/融合蛋白: | 3XFLAG(插入件上的N端子) |
克隆方法: | 限制性内切酶 |
5’克隆位点: | AgeI(未破坏) |
3’克隆位点: | EcoRI (未破坏) |
5’测序引物 | CTGGAGCACCTGCCTGAAATCACT |
文献引用方法 |
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材料和方法部分: | pSpCas9 (PX165) was a gift from Feng Zhang (Addgene plasmid # 48137 ; http://n2t.net/addgene:48137 ; RRID:Addgene_48137) |
参考文献部分: | Genome engineering using the CRISPR-Cas9 system. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. 10.1038/nprot.2013.143 PubMed 24157548 |
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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 pSpCas9 (PX165) 8214 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Human codon optimized Cas9 nuclease from Streptococcus pyogenes (Cbh-3X-FLAG-NLS-SpCas9-NLS). ACCESSION . VERSION . KEYWORDS pSpCas9 (PX165) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8214) AUTHORS Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F TITLE Genome engineering using the CRISPR-Cas9 system. JOURNAL Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. PUBMED 24157548 REFERENCE 2 (bases 1 to 8214) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8214 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 100..119 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(136..591) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(273..294) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 485..504 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 666..806 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 677..806 /label=AAV2 ITR polyA_signal 815..1022 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 1011..1028 /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" CDS complement(1056..1103) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS complement(1104..5204) /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS complement(5229..5249) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(5256..5321) /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" regulatory 5321..5330 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" intron 5342..5569 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" promoter complement(5570..5847) /label=chicken beta-actin promoter enhancer 5849..6134 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" repeat_region 6153..6282 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" rep_origin complement(6344..6932) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6424..6443) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7103..7963) /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 7726..7745 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7964..8068) /gene="bla" /label=AmpR promoter primer_bind 8136..8154 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(8192..8214) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 61 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 121 tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt 181 gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct tataaatcaa 241 aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt ccactattaa 301 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 361 gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 421 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 481 aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta gcggtcacgc 541 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tactatggtt 601 gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 661 gcgcccctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg 721 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 781 ggccaactcc atcactaggg gttcctgcgg ccgctcccca gcatgcctgc tattctcttc 841 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact 901 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc 961 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac 1021 agtcgaggct gatcagcgag ctctaggaat tcttactttt tcttttttgc ctggccggcc 1081 tttttcgtgg ccgccggcct tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg 1141 tacaggccgg tgatgctctg gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg 1201 tacctcttcc ggtcgatggt ggtgtcaaag tacttgaagg cggcaggggc tcccagattg 1261 gtcagggtaa acaggtggat gatattctcg gcctgctctc tgatgggctt atcccggtgc 1321 ttgttgtagg cggacagcac tttgtccaga ttagcgtcgg ccaggatcac tctcttggag 1381 aactcgctga tctgctcgat gatctcgtcc aggtagtgct tgtgctgttc cacaaacagc 1441 tgtttctgct cattatcctc gggggagccc ttcagcttct catagtggct ggccaggtac 1501 aggaagttca catatttgga gggcagggcc agttcgtttc ccttctgcag ttcgccggca 1561 gaggccagca ttctcttccg gccgttttcc agctcgaaca gggagtactt aggcagcttg 1621 atgatcaggt cctttttcac ttctttgtag cccttggctt ccagaaagtc gatgggattc 1681 ttctcgaagc tgcttctttc catgatggtg atccccagca gctctttcac actcttcagt 1741 ttcttggact tgcccttttc cactttggcc accaccagca cagaataggc cacggtgggg 1801 ctgtcgaagc cgccgtactt cttagggtcc cagtccttct ttctggcgat cagcttatcg 1861 ctgttcctct tgggcaggat agactctttg ctgaagccgc ctgtctgcac ctcggtcttt 1921 ttcacgatat tcacttgggg catgctcagc actttccgca cggtggcaaa atcccggccc 1981 ttatcccaca cgatctcccc ggtttcgccg tttgtctcga tcagaggccg cttccggatc 2041 tcgccgttgg ccagggtaat ctcggtcttg aaaaagttca tgatgttgct gtagaagaag 2101 tacttggcgg tagccttgcc gatttcctgc tcgctcttgg cgatcatctt ccgcacgtcg 2161 tacaccttgt agtcgccgta cacgaactcg ctttccagct tagggtactt tttgatcagg 2221 gcggttccca cgacggcgtt caggtaggcg tcgtgggcgt ggtggtagtt gttgatctcg 2281 cgcactttgt aaaactggaa atccttccgg aaatcggaca ccagcttgga cttcagggtg 2341 atcactttca cttcccggat cagcttgtca ttctcgtcgt acttagtgtt catccgggag 2401 tccaggatct gtgccacgtg ctttgtgatc tgccgggttt ccaccagctg tctcttgatg 2461 aagccggcct tatccagttc gctcaggccg cctctctcgg ccttggtcag attgtcgaac 2521 tttctctggg taatcagctt ggcgttcagc agctgccgcc agtagttctt catcttcttc 2581 acgacctctt cggagggcac gttgtcgctc ttgccccggt tcttgtcgct tctggtcagc 2641 accttgttgt cgatggagtc gtccttcaga aagctctgag gcacgatatg gtccacatcg 2701 tagtcggaca gccggttgat gtccagttcc tggtccacgt acatatcccg cccattctgc 2761 aggtagtaca ggtacagctt ctcgttctgc agctgggtgt tttccacggg gtgttctttc 2821 aggatctggc tgcccagctc tttgatgccc tcttcgatcc gcttcattct ctcgcggctg 2881 ttcttctgtc ccttctgggt ggtctggttc tctctggcca tttcgatcac gatgttctcg 2941 ggcttgtgcc ggcccatcac tttcacgagc tcgtccacca ccttcactgt ctgcaggatg 3001 cccttcttaa tggcggggct gccggccaga ttggcaatgt gctcgtgcag gctatcgccc 3061 tggccggaca cctgggcttt ctggatgtcc tctttaaagg tcaggctgtc gtcgtggatc 3121 agctgcatga agtttctgtt ggcgaagccg tcggacttca ggaaatccag gattgtcttg 3181 ccggactgct tgtcccggat gccgttgatc agcttccggc tcagcctgcc ccagccggtg 3241 tatctccgcc gcttcagctg cttcatcact ttgtcgtcga acaggtgggc ataggttttc 3301 agccgttcct cgatcatctc tctgtcctca aacagtgtca gggtcagcac gatatcttcc 3361 agaatgtcct cgttttcctc attgtccagg aagtccttgt ccttgataat tttcagcaga 3421 tcgtggtatg tgcccaggga ggcgttgaac cgatcttcca cgccggagat ttccacggag 3481 tcgaagcact cgattttctt gaagtagtcc tctttcagct gcttcacggt cactttccgg 3541 ttggtcttga acagcaggtc cacgatggcc tttttctgct cgccgctcag gaaggcgggc 3601 tttctcattc cctcggtcac gtatttcact ttggtcagct cgttatacac ggtgaagtac 3661 tcgtacagca ggctgtgctt gggcagcacc ttctcgttgg gcaggttctt atcgaagttg 3721 gtcatccgct cgatgaagct ctgggcggaa gcgcccttgt ccaccacttc ctcgaagttc 3781 cagggggtga tggtttcctc gctctttctg gtcatccagg cgaatctgct gtttcccctg 3841 gccagagggc ccacgtagta ggggatgcgg aaggtcagga tcttctcgat cttttcccgg 3901 ttgtccttca ggaatgggta aaaatcttcc tgccgccgca gaatggcgtg cagctctccc 3961 aggtggatct ggtgggggat gctgccgttg tcgaaggtcc gctgcttccg cagcaggtcc 4021 tctctgttca gcttcacgag cagttcctcg gtgccgtcca tcttttccag gatgggcttg 4081 atgaacttgt agaactcttc ctggctggct ccgccgtcaa tgtagccggc gtagccgttc 4141 ttgctctggt cgaagaaaat ctctttgtac ttctcaggca gctgctgccg cacgagagct 4201 ttcagcaggg tcaggtcctg gtggtgctcg tcgtatctct tgatcataga ggcgctcagg 4261 ggggccttgg tgatctcggt gttcactctc aggatgtcgc tcagcaggat ggcgtcggac 4321 aggttcttgg cggccagaaa caggtcggcg tactggtcgc cgatctgggc cagcaggttg 4381 tccaggtcgt cgtcgtaggt gtccttgctc agctgcagtt tggcatcctc ggccaggtcg 4441 aagttgctct tgaagttggg ggtcaggccc aggctcaggg caatcaggtt tccgaacagg 4501 ccattcttct tctcgccggg cagctgggcg atcagatttt ccagccgtct gctcttgctc 4561 agtctggcag acaggatggc cttggcgtcc acgccgctgg cgttgatggg gttttcctcg 4621 aacagctggt tgtaggtctg caccagctgg atgaacagct tgtccacgtc gctgttgtcg 4681 gggttcaggt cgccctcgat caggaagtgg ccccggaact tgatcatgtg ggccagggcc 4741 agatagatca gccgcaggtc ggccttgtcg gtgctgtcca ccagtttctt tctcaggtgg 4801 tagatggtgg ggtacttctc gtggtaggcc acctcgtcca cgatgttgcc gaagatgggg 4861 tgccgctcgt gcttcttatc ctcttccacc aggaaggact cttccagtct gtggaagaag 4921 ctgtcgtcca ccttggccat ctcgttgctg aagatctctt gcagatagca gatccggttc 4981 ttccgtctgg tgtatcttct tctggcggtt ctcttcagcc gggtggcctc ggctgtttcg 5041 ccgctgtcga acagcagggc tccgatcagg ttcttcttga tgctgtgccg gtcggtgttg 5101 cccagcacct tgaatttctt gctgggcacc ttgtactcgt cggtgatcac ggcccagccc 5161 acagagttgg tgccgatgtc caggccgatg ctgtacttct tgtcggctgc tgggactccg 5221 tggataccga ccttccgctt cttctttggg gccatcttat cgtcatcgtc tttgtaatca 5281 atatcatgat ccttgtagtc tccgtcgtgg tccttatagt ccatggtggc accggtccaa 5341 cctgaaaaaa agtgatttca ggcaggtgct ccaggtaatt aaacattaat accccaccaa 5401 ccaaccatcc cttaaaccct tacctcttgc tcagctaatt acagcccgga ggagaagggc 5461 cgtcccgccc gctcacctgt gggagtaacg cggtcagtca gagccggggc gggcggcgcg 5521 aggcggcggc ggagcggggc acggggcgaa ggcagcgcgc agcgactccc gcccgccgcg 5581 cgcttcgctt tttatagggc cgccgccgcc gccgcctcgc cataaaagga aactttcgga 5641 gcgcgccgct ctgattggct gccgccgcac ctctccgcct cgccccgccc cgcccctcgc 5701 cccgccccgc cccgcctggc gcgcgccccc cccccccccc cgcccccatc gctgcacaaa 5761 ataattaaaa aataaataaa tacaaaattg ggggtgggga ggggggggag atggggagag 5821 tgaagcagaa cgtggggctc acctcgacca tggtaatagc gatgactaat acgtagatgt 5881 actgccaagt aggaaagtcc cataaggtca tgtactgggc acaatgccag gcgggccatt 5941 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca 6001 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg 6061 ttactattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg 6121 taagttatgt aacgggtacc tctagaggcc gcaggaaccc ctagtgatgg agttggccac 6181 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 6241 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc 6301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 6361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 6421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 6481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 6541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 6601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 6661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 6721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 6781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 6841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 7021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 7081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 7141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 7201 gggagggctt accatctggc cccagtgctg caatgatacc gcggcttcca cgctcaccgg 7261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 7321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 7381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 7441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 7501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 7561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 7621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 7681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 7741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 7801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 7861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 7921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 7981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 8041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 8101 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 8161 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccgg //
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