pCas9大肠基因编辑质粒

北京庄盟国际生物基因科技有限公司 Beijing Zoman Biotechnology Co.,Ltd.pCas9大肠基因编辑质粒目录编号 产品名称 包装单位ZK1672 pCas9 1g(20l,50ng/l)储存: -20 。基本信息:复制子:p15A ori质粒分类:大肠系列质粒；大肠编辑质粒；大肠Cas9质粒质粒大小:9326bp原核抗性:氯霉素Chl（50g/ml）克隆菌株:Stbl3等大肠杆菌培养条件:37，LB，有氧表达宿主:大肠杆菌诱导方式:无需诱导5测序引物:StrepPyoCas9-5UTR-F（CGGTGCCACTTTTTCAAGTTG）3测序引物:pBRrevBam（GGTGATGTCGGCGATATAGG）质粒简介: pCas9质粒是CRISPR/Cas9系统类载体，复制子是p15A ori和ori，质粒大小是9326bp，可转化进相应的宿主细胞中，通大量养条件是LB，37。 pCas9可在大肠杆菌中表达Cas9蛋白，和pCRISPR搭配可用于基因敲除，低拷贝质粒，Bacterial expression of Cas9 nuclease, tracrRNA and crRNA guide. pCas9 can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-baseddirected evolution, and combinatorial design. CRISPR-Cas9 nucleases enable efficient genome editing in a wide variety of organisms and cell types . Target site recognition by Cas9 is programmed by a chimeric single guide RNA (sgRNA) that encodes a sequence complementary to a target protospacer5 , but also requires recognition of a short neighboring PAM . SpCas9, the most robust and widely used Cas9 to date, primarily recognizes NGG PAMs and is consequently restricted to sites that contain this motif . It can therefore be challenging to implement genome editing applications that require precision, such as: homology-directed repair (HDR), which is most efficient when DSBs are placed within 1020 bps of a desired alteration; the introduction of variable-length insertion or deletion (indel) mutations into small size genetic elements such as microRNAs, splice sites, short open reading frames, or transcription factor binding sites by non-homologous end-joining (NHEJ); and allele-specific editing, where PAM recognition might be exploited to differentiate alleles.质粒图谱:质粒序列:LOCUS Exported 9326 bp ds-DNA circular SYN 28-JUN-2017DEFINITION synthetic circular DNAKEYWORDS pCas9SOURCE synthetic DNA construct ORGANISM synthetic DNA constructREFERENCE 1 (bases 1 to 9326) TITLE Direct Submission JOURNAL Exported Wednesday, June 28, 2017 from MiaoLingPlasmid FEATURES Location/Qualifiers source 1.9326 /organism=synthetic DNA construct /mol_type=other DNA promoter complement(220.322) /note=cat promoter /note=promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase rep_origin complement(848.1393) /direction=LEFT /note=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. promoter 1505.1533 /note=tet promoter /note=E. coli promoter for tetracycline efflux protein gene misc_RNA complement(1851.1929) /note=tracrRNA /note=trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system CDS 2225.6331 /codon_start=1 /product=Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system /note=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 misc_feature 6352.6483 /note=crRNA leader /note=crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system repeat_region 6484.6519 /note=DR /note=direct repeat for the Streptococcus pyogenes CRISPR/Cas system repeat_region 6550.6585 /note=DR /note=direct repeat for the Streptococcus pyogenes CRISPR/Cas system CDS complement(8886.219) /codon_start=1 /gene=cat /product=chloramphenicol acetyltransferase /note=CmR /note=confers resistance to chloramphenicol /translation=MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGAORIGIN 1 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt 61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt 121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga 181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga 241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt 301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc 361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat 421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt 481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg 541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact 601 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa 661 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc 721 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc 781 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa 841 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc 901 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc 961 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc 1021 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 1081 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 1141 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt 1201 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 1261 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 1321 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 1381 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca 1441 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc 1501 atgtttgaca gcttatcatc gataagcttt aatgcggtag tttatcacag ttaaattgct 1561 aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc ctcggcaccg 1621 tcaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc ctcttgcggg 1681 attacgaaat catcctgtgg agcttagtag gtttagcaag atggcagcgc ctaaatgtag 1741 aatgataaaa ggattaagag attaatttcc ctaaaaatga taaaacaagc gttttgaaag 1801 cgcttgtttt tttggtttgc agtcagagta gaatagaagt atcaaaaaaa gcaccgactc 1861 ggtgccactt tttcaagttg ataacggact agccttattt taacttgcta tgctgttttg 1921 aatggttcca acaagattat tttataactt ttataacaaa taatcaagga gaaattcaaa 1981 gaaatttatc agccataaaa caatacttaa tactatagaa tgataacaaa ataaactact 2041 ttttaaaaga attttgtgtt ataatctatt tattattaag tattgggtaa tattttttga 2101 agagatattt tgaaaaagaa aaattaaagc atattaaact aatttcggag gtcattaaaa 2161 ctattattga aatcatcaaa ctcattatgg atttaattta aactttttat tttaggaggc 2221 aaaaatggat aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc 2281 ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga 2341 ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc 2401 ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat 2461 ttgttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca 2521 tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt 2581 tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg 2641 aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc 2701 gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag 2761 tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa 2821 ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc 2881 aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg 2941 gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc 3001 agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt 3061 ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc 3121 tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc 3181 ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt 3241 tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata 3301 tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat 3361 tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct 3421 gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact tgggtgagct 3481 gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa 3541 gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa 3601 tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga 3661 agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga 3721 taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac 3781 ggtttataac gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt 3841 tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt 3901 aaccgttaag caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga 3961 aatttcagga gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa 4021 aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat 4081 tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata 4141 tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg 4201 gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat 4261 attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga 4321 tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag 4381 tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca 4441 gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat 4501 cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga 4561 gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca 4621 tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg 4681 aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga 4741 tcacattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg 4801 ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat 4861 gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa 4921 tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg 4981 ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat 5041 gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa 5101 atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa 5161 caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa 5221 gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg 5281 taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta 5341 ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa 5401 acgccctcta atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga 5461 ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga 5521 agtacagaca ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct 5581 tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt 5641 agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc 5701 cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat 5761 tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc 5821 taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga 5881 attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc 5941 tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt 6001 ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg 6061 tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga 6121 caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg 6181 agctcccgct gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac 6241 aaaagaagtt ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg 6301 cattgatttg agtcagctag gaggtgactg aagtatattt tagatgaaga ttatttctta 6361 ataactaaaa atatggtata atactcttaa taaatgcagt aatacagggg cttttcaaga 6421 ctgaagtcta gctgagacaa atagtgcgat tacgaaattt tttagacaaa aatagtctac 6481 gaggttttag agctatgctg ttttgaatgg tcccaaaact gagaccagtc tcggaagctc 6541 aaaggtctcg ttttagagct atgctgtttt gaatggtccc aaaacttcag cacactgaga 6601 cttgttgagt tccatgtttt agagctatgc tgttttgaat ggactccatt caacattgcc 6661 gatgataact tgagaaagag ggttaatacc agcagtcgga taccttccta ttctttctgt 6721 taaagcgttt tcatgttata ataggcaaaa gaagagtagt gtgatcgtcc attccgacag 6781 catcgccagt cactatggcg tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc 6841 acccgttctc ggagcactgt ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct 6901 acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt ggatcctcta 6961 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat 7021 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt 7081 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca 7141 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct 7201 aatgcaggag tcgcataagg gagagcgtcg accgatgccc ttgagagcct tcaacccagt 7261 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt 7321 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg 7381 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc 7441 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat 7501 tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg 7561 ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga tgcccgcgtt 7621 gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc aaggatcgct 7681 cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg cgatttatgc 7741 cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt 7801 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc 7861 cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg 7921 agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc 7981 agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc 8041 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat 8101 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga 8161 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtcc 8221 cctacgtgct gctgaagttg cccgcaacag agagtggaac caaccggtga taccacgata 8281 ctatgactga gagtcaacgc catgagcggc ctcatttctt attctgagtt acaacagtcc 8341 gcaccgctgt ccggtagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 8401 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgcc caacagtccc 8461 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgccctgcac cattatgttc 8521 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg 8581 aagcgctaac cgtttttatc aggctctggg aggcagaata aatgatcata tcgtcaatta 8641 ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag cacacggtca 8701 cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac 8761 gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg 8821 cttattatca cttattcagg cgtagcacca ggcgtttaag ggcaccaata actgccttaa 8881 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 8941 ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg catcagcacc 9001 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata 9061 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 9121 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 9181 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 9241 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 9301 agctcaccgt ctttcattgc 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