质粒名称: | ABE9 |
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出品公司: | Addgene |
目录编号: | 194208 |
存储实验室: | Dali Li |
载体骨架基本信息 |
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载体名称: | pCMV |
完整质粒大小: | 8217 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: | DH5a |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | TadA-8e (N108Q/L145T)-nSpCas9 |
插入大小: | 4818bp |
标签/融合蛋白: | 6*His (C terminal on backbone) |
克隆方法: | Gibson Cloning |
5’测序引物 | CMV-Forward |
3’测序引物 | BGH-Reverse |
文献引用方法 |
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材料和方法部分: | ABE9 was a gift from Dali Li (Addgene plasmid # 194208 ; http://n2t.net/addgene:194208 ; RRID:Addgene_194208) |
参考文献部分: | Engineering a precise adenine base editor with minimal bystander editing. Chen L, Zhang S, Xue N, Hong M, Zhang X, Zhang D, Yang J, Bai S, Huang Y, Meng H, Wu H, Luan C, Zhu B, Ru G, Gao H, Zhong L, Liu M, Liu M, Cheng Y, Yi C, Wang L, Zhao Y, Song G, Li D. Nat Chem Biol. 2022 Oct 13. pii: 10.1038/s41589-022-01163-8. doi: 10.1038/s41589-022-01163-8. 10.1038/s41589-022-01163-8 PubMed 36229683 |
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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 ABE9 8217 bp ds-DNA circular SYN 10-JAN-2023 DEFINITION Mammalian expression of ABE9. ACCESSION . VERSION . KEYWORDS ABE9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8217) AUTHORS Chen L, Zhang S, Xue N, Hong M, Zhang X, Zhang D, Yang J, Bai S, Huang Y, Meng H, Wu H, Luan C, Zhu B, Ru G, Gao H, Zhong L, Liu M, Liu M, Cheng Y, Yi C, Wang L, Zhao Y, Song G, Li D TITLE Engineering a precise adenine base editor with minimal bystander editing. JOURNAL Nat Chem Biol. 2022 Oct 13. pii: 10.1038/s41589-022-01163-8. doi: 10.1038/s41589-022-01163-8. PUBMED 36229683 REFERENCE 2 (bases 1 to 8217) AUTHORS . TITLE Direct Submission JOURNAL Exported Jan 10, 2023 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..8217 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 286..306 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 377..396 /label=T7 /note="T7 promoter, forward primer" promoter 377..395 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 445..465 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1060..5160 /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5203..5223 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5232..5249 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(5272..5289) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5278..5502 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(5573..5589) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5573..5589) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5586..5608) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5597..5613 /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(5621..5651) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5666..5687 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5804..5821) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5975..6563) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6055..6074) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6734..7594) /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 7357..7376 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7595..7699) /gene="bla" /label=AmpR promoter primer_bind complement(7778..7797) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 7969..131 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt 481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg 541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac 601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc 661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct 721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg 781 aggcagtcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg 841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgacctgc 901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc 961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc 1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcag acaagaagta cagcatcggc 1081 ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc 1141 agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc 1201 ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc 1261 agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac 1321 gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa 1381 gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac 1441 cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag 1501 gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc 1561 ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg 1621 gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc 1681 aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag 1741 ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg 1801 acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag 1861 gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac 1921 ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg 1981 aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac 2041 caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa 2101 gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc 2161 caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa 2221 ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc 2281 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat 2341 ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc 2401 ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag 2461 agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc 2521 cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg 2581 cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa 2641 tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc 2701 gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac 2761 ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac 2821 gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac 2881 aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac 2941 agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg 3001 aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac 3061 ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc 3121 gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc 3181 cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc 3241 ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg 3301 aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag 3361 accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc 3421 aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac 3481 gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg 3541 gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag 3601 gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac 3661 aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac 3721 gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg 3781 agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca 3841 aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag 3901 ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag 3961 gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac 4021 ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc 4081 gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa 4141 atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc 4201 gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa 4261 accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc 4321 atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag 4381 tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct 4441 aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa 4501 gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc 4561 atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa 4621 gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac 4681 ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg 4741 ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc 4801 cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag 4861 atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac 4921 aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat 4981 atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac 5041 accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc 5101 caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggtgac 5161 tctggcggct caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa 5221 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg 5281 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 5341 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 5401 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 5461 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 5521 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc 5581 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 5641 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 5701 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 5761 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 5821 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 5881 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 5941 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6001 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6061 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6121 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6181 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6241 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6301 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 6361 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 6421 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 6481 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 6541 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc 6601 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 6661 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 6721 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 6781 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 6841 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 6901 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 6961 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7021 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7081 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7141 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7201 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7261 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7321 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 7381 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 7441 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 7501 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 7561 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 7621 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 7681 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg 7741 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat 7801 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca 7861 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg 7921 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat 7981 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatattgagt 8041 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8101 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8161 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc //
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