| 质粒名称: | pEM-Cas9HF1-recA56 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 89962 |
| 存储实验室: | Michael Lynch |
|
载体骨架基本信息 |
|
| 载体名称: | pEM-Cas9HF1 |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 7859 bp |
| 载体修饰: | 人细胞色素c氧化酶亚基VIII（human cytochrome C oxidase subunit VIII） |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9HF1 |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | AgeI(未破坏) |
| 3' 克隆位点: | NotI (未破坏) |
| 5' 测序引物 | CMV-F |
| 3' 测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: | pEM-Cas9HF1-recA56 was a gift from Michael Lynch (Addgene plasmid # 89962 ; http://n2t.net/addgene:89962 ; RRID:Addgene_89962) |
| 参考文献部分: | Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity. Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar R, Lynch MD. ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174. 10.1021/acssynbio.7b00174 PubMed 28915012 |
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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 pEM-Cas9HF1-recA56 7953 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Modified from pEM-Cas9HF1 (Addgene ID: 89961) to include constitutive recA56 to block recA-mediated double-strand break repair.. ACCESSION . VERSION . KEYWORDS pEM-Cas9HF1-recA56 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7953) AUTHORS Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar R, Lynch MD TITLE Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity. JOURNAL ACS Synth Biol. 2017 Oct 2. doi: 10.1021/acssynbio.7b00174. PUBMED 28915012 REFERENCE 2 (bases 1 to 7953) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7953 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(10..633) /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind 541..560 /label=Tet-R /note="Tetracycline resistance gene, reverse primer" promoter 684..739 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 720..738 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 775..4878 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Kleinstiver et al., 2016)" /label=SpCas9-HF1 /note="carries the mutations N497A, R661A, Q695A, and Q926A" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFM ALIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5030..6091 /codon_start=1 /gene="recA" /product="DNA repair protein from E. coli" /label=RecA /translation="MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGS LSLDIALGAGGLPMGCIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIY ARKLGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDS HMGLAARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASV RLDIRRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVK EKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSV DDSEGVAETNEDF" terminator 6121..6192 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6208..6235 /label=T7Te terminator /note="phage T7 early transcription terminator" primer_bind complement(6263..6280) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6397..6942) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(6477..6496) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 7056..7150 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(7171..7830) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 7745..7764 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(7831..7933) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" ORIGIN 1 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa 61 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg 121 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg 181 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata 241 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt 301 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact 361 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc 421 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag 481 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg 541 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct 601 gttaatcact ttacttttat ctaatctaga catcattaat tcctaattgc tagcattgta 661 cctaggactg agctagccat aaagttgaca ctctatcgtt gatagagtta ttttaccact 721 ccctatcagt gatagagaaa agaattcaaa agatctaaag aggacttcgg atctatggat 781 aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc ggtgatcact 841 gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga ccgccacagt 901 atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc ggaagcgact 961 cgcctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat ttgttatcta 1021 caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca tcgacttgaa 1081 gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt tggaaatata 1141 gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg aaaaaaattg 1201 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt 1261 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac 1321 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac 1381 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta 1441 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt 1501 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct 1561 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt 1621 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt 1681 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt 1741 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa 1801 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat 1861 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa 1921 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa 1981 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt 2041 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa 2101 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt 2161 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc 2221 gataaaggtg cttcagctca atcatttatt gaacgcatga cagcgtttga taaaaatctt 2281 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac 2341 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt 2401 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag 2461 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga 2521 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa 2581 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca 2641 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc 2701 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggagcgttg 2761 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt 2821 ttgaaatcag atggttttgc caatcgcaat tttatggcgc tgatccatga tgatagtttg 2881 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa 2941 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa 3001 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa 3061 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa 3121 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa 3181 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg 3241 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tcacattgtt 3301 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa 3361 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat 3421 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa 3481 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt 3541 gaaactcgcg cgatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa 3601 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta 3661 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat 3721 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca 3781 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt 3841 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc 3901 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta 3961 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca 4021 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca 4081 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt 4141 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca 4201 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag 4261 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta 4321 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt 4381 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa 4441 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat 4501 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat 4561 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta 4621 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata 4681 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct 4741 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt 4801 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg 4861 agtcagctag gaggtgacgc tgctaacgac gaaaactacg ctctggccgc ctaatattac 4921 ccggcatgac atttacgggc atgcataagg ctcgtataat atattcaggg agaccacaac 4981 ggtttccctc tacaaataat tttgtttaac tttcatgaca ggagtaaaaa tggctatcga 5041 cgaaaacaaa cagaaagcgt tggcggcagc actgggccag attgagaaac aatttggtaa 5101 aggctccatc atgcgcctgg gtgaagaccg ttccatggat gtggaaacca tctctaccgg 5161 ttcgctttca ctggatatcg cgcttggggc aggtggtctg ccgatgggct gcatcgtcga 5221 aatctacgga ccggaatctt ccggtaaaac cacgctgacg ctgcaggtga tcgccgcagc 5281 gcagcgtgaa ggtaaaacct gtgcgtttat cgatgctgaa cacgcgctgg acccaatcta 5341 cgcacgtaaa ctgggcgtcg atatcgacaa cctgctgtgc tcccagccgg acaccggcga 5401 gcaggcactg gaaatctgtg acgccctggc gcgttctggc gcagtagacg ttatcgtcgt 5461 tgactccgtg gcggcactga cgccgaaagc ggaaatcgaa ggcgaaatcg gcgactctca 5521 catgggcctt gcggcacgta tgatgagcca ggcgatgcgt aagctggcgg gtaacctgaa 5581 gcagtccaac acgctgctga tcttcatcaa ccagatccgt atgaaaattg gtgtgatgtt 5641 cggtaacccg gaaaccacta ccggtggtaa cgcgctgaaa ttctacgcct ctgttcgtct 5701 ggacatccgt cgtatcggcg cggtgaaaga gggcgaaaac gtggtgggta gcgaaacccg 5761 cgtgaaagtg gtgaagaaca aaatcgctgc gccgtttaaa caggctgaat tccagatcct 5821 ctacggcgaa ggtatcaact tctacggcga actggttgac ctgggcgtaa aagagaagct 5881 gatcgagaaa gcaggcgcgt ggtacagcta caaaggtgag aagatcggtc agggtaaagc 5941 gaatgcgact gcctggctga aagataaccc ggaaaccgcg aaagagatcg agaagaaagt 6001 acgtgagttg ctgctgagca acccgaactc aacgccggat ttctctgtag atgatagcga 6061 aggcgtagca gaaactaacg aagattttta agcttaactc gagtaaggat ctccaggcat 6121 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 6181 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat 6241 acctagggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc 6301 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac 6361 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga 6421 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag 6481 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt 6541 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt 6601 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc 6661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac 6721 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt 6781 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg 6841 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg 6901 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat 6961 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc 7021 agccccatac gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg 7081 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc 7141 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta 7201 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac 7261 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa 7321 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac 7381 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag 7441 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc 7501 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca 7561 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg 7621 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt 7681 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca 7741 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac 7801 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga 7861 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct 7921 tacgtgccga tcaacgtctc attttcgcca gat //
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