质粒名称: | pCMV-T7-SpCas9-P2A-EGFP (RTW3027) |
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出品公司: | Addgene |
目录编号: | 139987 |
存储实验室: | Benjamin Kleinstiver |
载体骨架基本信息 |
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载体名称: | JDS246 |
载体出品公司: | Keith Joung (Addgene Plasmid 43861) |
空载体大小: | -- |
完整质粒大小: | 8451 bp |
载体修饰: | -- |
载体类型: | 哺乳动物表达;体外转录;T7启动子 |
筛选标记: | 新霉素(G418) |
载体抗性: |
氨苄青霉素,100μg/mL
|
生长温度: | 37 ℃ |
宿主菌: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | 用BPNLS-3xFLAG-P2A-EGFP优化人密码子SpCas9 |
物种来源: | Synthetic |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子: | CMV and T7 |
标签/融合蛋白: | BPNLS-3xFLAG-P2A-EGFP(插入物上的C端) |
克隆方法: | Gibson Cloning |
5’克隆位点: | -- |
3’克隆位点: | -- |
5’测序引物 | oBK6928-CCAAGTCTCCACCCCATTGACG |
3’测序引物 | oBK219-GGGAGTGGCACCTTCCAGGGTC |
文献引用方法 |
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材料和方法部分: | pCMV-T7-SpCas9-P2A-EGFP (RTW3027) was a gift from Benjamin Kleinstiver (Addgene plasmid # 139987 ; http://n2t.net/addgene:139987 ; RRID:Addgene_139987) |
参考文献部分: | Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Walton RT, Christie KA, Whittaker MN, Kleinstiver BP. Science. 2020 Apr 17;368(6488):290-296. doi: 10.1126/science.aba8853. Epub 2020 Mar 26. 10.1126/science.aba8853 PubMed 32217751 |
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载体基本信息
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载体简介
载体序列
LOCUS pCMV-T7-SpCas9-P2A-EGFP (RTW3027) 8451 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CMV and T7 promoter expression plasmid for human codon optimized SpCas9 with a c-terminal bi-partite NLS, 3x flag tag, and P2A-EGFP. ACCESSION . VERSION . KEYWORDS pCMV-T7-SpCas9-P2A-EGFP (RTW3027) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8451) AUTHORS Walton RT, Christie KA, Whittaker MN, Kleinstiver BP TITLE Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. JOURNAL Science. 2020 Apr 17;368(6488):290-296. doi: 10.1126/science.aba8853. Epub 2020 Mar 26. PUBMED 32217751 REFERENCE 2 (bases 1 to 8451) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..8451 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 3992..4012 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4025..4090 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4109..4165 /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" CDS 4166..4882 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4211..4232) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4472..4491) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4819..4840 /label=EGFP-C /note="EGFP, forward primer" CDS 4891..4908 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4931..4948) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4937..5161 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5232..5248) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5232..5248) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5245..5267) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5256..5272 /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)." promoter complement(5280..5310) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5325..5346 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5463..5480) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5634..6222) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5714..5733) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6393..7253) /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 7016..7035 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7254..7358) /gene="bla" /label=AmpR promoter primer_bind complement(7437..7456) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 7628..8007 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 8008..8211 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 8162..8182 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 8253..8272 /label=T7 /note="T7 promoter, forward primer" promoter 8253..8271 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 8279..8288 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 8285..3937 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" ORIGIN 1 cgaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa 61 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag 121 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca 181 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta 241 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct 301 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc 361 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt 421 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact 481 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg 541 cctgttcgga aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt 601 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga 661 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct 721 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc 781 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa 841 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa 901 gaacggctac gccggctaca ttgacggcgg agccagccag gaagagttct acaagttcat 961 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga 1021 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct 1081 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa 1141 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc 1201 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg 1261 gaacttcgag gaagtggtgg acaagggcgc ttccgcccag agcttcatcg agcggatgac 1321 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga 1381 gtacttcacc gtgtataacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa 1441 gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa 1501 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga 1561 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga 1621 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct 1681 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct 1741 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata 1801 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg 1861 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct 1921 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca 1981 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg 2041 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc 2101 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa 2161 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct 2221 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct 2281 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta 2341 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg acaacaaggt 2401 gctgaccaga agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt 2461 gaagaagatg aagaactact ggcggcagct gctgaacgcc aagctgatta cccagagaaa 2521 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt 2581 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga 2641 ctcccggatg aacactaagt acgacgagaa tgacaagctg atccgggaag tgaaagtgat 2701 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg 2761 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc 2821 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta 2881 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta 2941 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga 3001 gatccggaag cggcctctga tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa 3061 gggccgggat tttgccaccg tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa 3121 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag 3181 cgataagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag 3241 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa 3301 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa 3361 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat 3421 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc 3481 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct 3541 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca 3601 gctgtttgtg gaacagcaca agcactacct ggacgagatc atcgagcaga tcagcgagtt 3661 ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa 3721 gcaccgggat aagcccatca gagagcaggc cgagaatatc atccacctgt ttaccctgac 3781 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta 3841 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta 3901 cgagacacgg atcgacctgt ctcagctggg aggcgacgga tccggcggag gcggaagcgg 3961 gaaaagaacc gccgacggca gcgaattcga gcccaagaag aagaggaaag tctcgagcgg 4021 aggcgactac aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga 4081 cgatgacaag ggaggcggtg gaagcggcgc aacaaacttc tctctgctga aacaagccgg 4141 agatgtcgaa gagaatcctg gaccgatggt gagcaagggc gaggagctgt tcaccggggt 4201 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg 4261 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg 4321 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt 4381 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg 4441 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga 4501 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa 4561 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta 4621 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat 4681 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg 4741 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc 4801 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct 4861 cggcatggac gagctgtaca agtgaccggt catcatcacc atcaccattg agtttaaacc 4921 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 4981 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 5041 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 5101 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 5161 gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg 5221 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 5281 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc 5341 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 5401 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 5461 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 5521 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 5581 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5641 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5701 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5761 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5821 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5881 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5941 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 6001 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 6061 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 6121 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 6181 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 6241 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 6301 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 6361 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 6421 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 6481 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 6541 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 6601 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 6661 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 6721 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 6781 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 6841 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 6901 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6961 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 7021 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 7081 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 7141 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 7201 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 7261 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 7321 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 7381 cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca 7441 atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 7501 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 7561 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 7621 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 7681 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 7741 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 7801 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 7861 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 7921 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 7981 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 8041 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 8101 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 8161 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg 8221 aaccgtcaga tccgctagag atccgcggcc gctaatacga ctcactatag ggagagccgc 8281 caccatggac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc 8341 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga 8401 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg g //
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