质粒名称: | pCRISPRia-v2 |
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出品公司: | 爱迪基因 |
目录编号: | 84832 |
存储实验室: | Jonathan Weissman |
载体骨架基本信息 |
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载体名称: | pSICO derivative |
载体出品公司: | Addgene |
载体抗性: | 氨苄青霉素 |
空载体大小: | 8200 bp |
完整质粒大小: | 8888bp |
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 |
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插入基因名称: | sgRNA |
物种来源: | -- |
插入大小: | 113 bp |
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 |
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材料和方法部分: | pCRISPRia-v2 was a gift from Jonathan Weissman (Addgene plasmid # 84832 ; http://n2t.net/addgene:84832 ; RRID:Addgene_84832) |
参考文献部分: | Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, Fields AP, Park CY, Corn JE, Kampmann M, Weissman JS. Elife. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760. 10.7554/eLife.19760 PubMed 27661255 |
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载体基本信息
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载体序列
LOCUS pCRISPRia-v2 8893 bp ds-DNA circular SYN 22-APR-2019 DEFINITION CRISPRi/a V2 library parental plasmid. ACCESSION . VERSION . KEYWORDS pCRISPRia-v2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8893) AUTHORS Horlbeck MA, Gilbert LA, Villalta JE, Adamson B, Pak RA, Chen Y, Fields AP, Park CY, Corn JE, Kampmann M, Weissman JS TITLE Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. JOURNAL Elife. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760. PUBMED 27661255 REFERENCE 2 (bases 1 to 8893) AUTHORS . TITLE Direct Submission JOURNAL Exported Apr 22, 2019 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8893 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1684..1917 /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 2444..2561 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2822..2855 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 2859..2879 /label=mU6-F /note="Mouse U6 promoter, forward primer" promoter 3132..3343 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" intron 3344..4282 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4239..4259 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 4303..4899 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(4303..4322) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4799..4819 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 4909..4962 /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 4975..5673 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" protein_bind 5693..5726 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5782..6370 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5835..5855) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6253..6264) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6373..6389) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6374..6390) /label=pBluescriptKS /note="For pBluescript vector" LTR 6899..7079 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(7141..7729) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7221..7240) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7900..8760) /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 8523..8542 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8761..8865) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga 1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt 1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg 1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc 1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa 1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa 1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg 1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg 1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga 2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga 2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa 2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg 2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc 2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga 2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact 2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg 2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa 2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca 2581 gcagagatcc agtttggtta gtaccgggcc cgccctgcag gtctagagat ccgacgcgcc 2641 atctctaggc ccgcgccggc cccctcgcac ggacttgtgg gagaagctcg gctactcccc 2701 tgccccggtt aatttgcata taatatttcc tagtaactat agaggcttaa tgtgcgataa 2761 aagacagata atctgttctt tttaatacta gctacatttt acatgatagg cttggatttc 2821 tataacttcg tatagcatac attatacgaa gttataaaca gcacaaaagg aaactcaccc 2881 taactgtaaa gtaattgtgt gttttgagac tataagtatc ccttggagaa ccaccttgtt 2941 ggaccaggat gggcaccacc cgtttaagag ctaagctgga aacagcatag caagtttaaa 3001 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttctcgag 3061 tactaggatc cattaggcgg ccgccctgca gggtggataa ccgtattacc gccatgcatg 3121 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt 3181 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt 3241 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc 3301 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg 3361 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac 3421 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc 3481 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg 3541 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa 3601 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag 3661 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg 3721 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc 3781 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc 3841 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg 3901 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg 3961 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc 4021 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc 4081 gcgcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc 4141 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt 4201 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca 4261 aagttttttt cttccatttc aggtgtcgtg agctagccca ccatgaccga gtacaagccc 4321 acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg 4381 ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc 4441 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 4501 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 4561 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 4621 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 4681 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 4741 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 4801 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 4861 ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatcgggaga gggcagagga 4921 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caccggtcgc caccagcgag 4981 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac 5041 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc 5101 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc 5161 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc 5221 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc 5281 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg 5341 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc 5401 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag 5461 ctcgtgggcg ggagccatct gatcgcaaac atcaagacca catatagatc caagaaaccc 5521 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag 5581 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac 5641 ctccctagca aactggggca caagcttaat taagaattcg tcgagggacc taataacttc 5701 gtatagcata cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa 5761 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact 5821 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg 5881 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg 5941 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg 6001 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg 6061 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc 6121 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca 6181 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc 6241 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct 6301 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc 6361 gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac 6421 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga 6481 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc 6541 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca 6601 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg 6661 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa 6721 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt 6781 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag 6841 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg 6901 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 6961 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 7021 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 7081 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 7141 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 7201 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 7261 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 7321 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 7381 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 7441 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 7501 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 7561 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 7621 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 7681 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 7741 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 7801 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 7861 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 7921 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 7981 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 8041 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 8101 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 8161 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 8221 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 8281 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 8341 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 8401 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 8461 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 8521 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 8581 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 8641 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 8701 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 8761 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 8821 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 8881 agtgccacct gac //
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