LOCUS Exported 6388 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION AtDeadCAS9 D10A H840A module CDS1. ACCESSION . VERSION . KEYWORDS pORFE0003 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6388) TITLE Chevalier Lab plasmids REFERENCE 2 (bases 1 to 6388) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6388 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 2064..2084 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(2127..2145) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2213..2317 /gene="bla" /label=AmpR promoter CDS 2537..3328 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 3499..4087 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3988..4007 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4241..4258 /label=L4440 /note="L4440 vector, forward primer" CDS 4336..2051 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" ORIGIN 1 ctgatggatt cgctaacaga aacttcatgc agctcatcca cgatgattct ctcaccttta 61 aagaggatat ccagaaggct caggtttcag gacagggtga tagtctccat gagcatatcg 121 ctaacctcgc tggatctcct gcaatcaaga agggaatcct ccagactgtg aaggttgtgg 181 atgagttggt gaaggtgatg ggaaggcata agcctgagaa catcgtgatc gaaatggcta 241 gagagaacca gaccactcag aagggacaga agaactctag ggaaaggatg aagaggatcg 301 aggaaggtat caaagagctt ggatctcaga tcctcaaaga gcaccctgtt gagaacactc 361 agctccagaa tgagaagctc tacctctact acctccagaa cggaagggat atgtatgtgg 421 atcaagagtt ggatatcaac aggctctctg attacgatgt tgatgctatc gtgccacagt 481 cattcttgaa ggatgattct atcgataaca aggtgctcac caggtctgat aagaacaggg 541 gtaagagtga taacgtgcca agtgaagagg ttgtgaagaa aatgaagaac tattggaggc 601 agctcctcaa cgctaagctc atcactcaga gaaagttcga taacttgact aaggctgaga 661 ggggaggact ctctgaattg gataaggcag gattcatcaa gaggcagctt gtggaaacca 721 ggcagatcac taagcacgtt gcacagatcc tcgattctag gatgaacacc aagtacgatg 781 agaacgataa gttgatcagg gaagtgaagg ttatcaccct caagtcaaag ctcgtgtctg 841 atttcagaaa ggatttccaa ttctacaagg tgagggaaat caacaactac caccacgctc 901 acgatgctta ccttaacgct gttgttggaa ccgctctcat caagaagtat cctaagctcg 961 agtcagagtt cgtgtacggt gattacaagg tgtacgatgt gaggaagatg atcgctaagt 1021 ctgagcaaga gatcggaaag gctaccgcta agtatttctt ctactctaac atcatgaatt 1081 tcttcaagac cgagattacc ctcgctaacg gtgagatcag aaagaggcca ctcatcgaga 1141 caaacggtga aacaggtgag atcgtgtggg ataagggaag ggatttcgct accgttagaa 1201 aggtgctctc tatgccacag gtgaacatcg ttaagaaaac cgaggtgcag accggtggat 1261 tctctaaaga gtctatcctc cctaagagga actctgataa gctcattgct aggaagaagg 1321 attgggaccc taagaaatac ggtggtttcg attctcctac cgtggcttac tctgttctcg 1381 ttgtggctaa ggttgagaag ggaaagagta agaagctcaa gtctgttaag gaacttctcg 1441 gaatcactat catggaaagg tcatctttcg agaagaaccc aatcgatttc ctcgaggcta 1501 agggatacaa agaggttaag aaggatctca tcatcaagct cccaaagtac tcactcttcg 1561 aactcgagaa cggtagaaag aggatgctcg cttctgctgg tgagcttcaa aagggaaacg 1621 agcttgctct cccatctaag tacgttaact ttctttacct cgcttctcac tacgagaagt 1681 tgaagggatc tccagaagat aacgagcaga agcaactttt cgttgagcag cacaagcact 1741 acttggatga gatcatcgag cagatctctg agttctctaa aagggtgatc ctcgctgatg 1801 caaacctcga taaggtgttg tctgcttaca acaagcacag agataagcct atcagggaac 1861 aggcagagaa catcatccat ctcttcaccc ttaccaacct cggtgctcct gctgctttca 1921 agtacttcga tacaaccatc gataggaaga gatacacctc taccaaagaa gtgctcgatg 1981 ctaccctcat ccatcagtct atcactggac tctacgagac taggatcgat ctctcacagc 2041 tcggtggtga ttcaagggct gatcctaaga agaagaggaa ggtttgagct ttgagaccac 2101 gaagtggctc ttcagtggac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 2161 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 2221 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 2281 ctgataaatg cttcaataat attgaaaaag gaagagtatg cgctcacgca actggtccag 2341 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga 2401 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt 2461 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa 2521 aacaaagtta aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga 2581 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg 2641 ctccgcagtg gatggcggcc tgaagccaca cagcgatatt gatttgctgg ttacggtgac 2701 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc 2761 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga 2821 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa 2881 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct 2941 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga 3001 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc 3061 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta 3121 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg 3181 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga 3241 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccatt acgtaaaagg 3301 cgagatcacc aaggtagtcg gcaaataact gtcagaccaa gtttactcat atatacttta 3361 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 3421 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 3481 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 3541 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 3601 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 3661 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 3721 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 3781 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 3841 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 3901 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 3961 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 4021 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 4081 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 4141 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 4201 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 4261 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatc 4321 actctgtggt ctcaaatgga taagaagtac tctatcggac tcgctatcgg aactaactct 4381 gtgggatggg ctgtgatcac cgatgagtac aaggtgccat ctaagaagtt caaggttctc 4441 ggaaacaccg ataggcactc tatcaagaaa aaccttatcg gtgctctcct cttcgattct 4501 ggtgaaactg ctgaggctac cagactcaag agaaccgcta gaagaaggta caccagaaga 4561 aagaacagga tctgctacct ccaagagatc ttctctaacg agatggctaa agtggatgat 4621 tcattcttcc acaggctcga agagtcattc ctcgtggaag aagataagaa gcacgagagg 4681 caccctatct tcggaaacat cgttgatgag gtggcatacc acgagaagta ccctactatc 4741 taccacctca gaaagaagct cgttgattct actgataagg ctgatctcag gctcatctac 4801 ctcgctctcg ctcacatgat caagttcaga ggacacttcc tcatcgaggg tgatctcaac 4861 cctgataact ctgatgtgga taagttgttc atccagctcg tgcagaccta caaccagctt 4921 ttcgaagaga accctatcaa cgcttcaggt gtggatgcta aggctatcct ctctgctagg 4981 ctctctaagt caagaaggct tgagaacctc attgctcagc tccctggtga gaagaagaac 5041 ggacttttcg gaaacttgat cgctctctct ctcggactca cccctaactt caagtctaac 5101 ttcgatctcg ctgaggatgc aaagctccag ctctcaaagg atacctacga tgatgatctc 5161 gataacctcc tcgctcagat cggagatcag tacgctgatt tgttcctcgc tgctaagaac 5221 ctctctgatg ctatcctcct cagtgatatc ctcagagtga acaccgagat caccaaggct 5281 ccactctcag cttctatgat caagagatac gatgagcacc accaggatct cacacttctc 5341 aaggctcttg ttagacagca gctcccagag aagtacaaag agattttctt cgatcagtct 5401 aagaacggat acgctggtta catcgatggt ggtgcatctc aagaagagtt ctacaagttc 5461 atcaagccta tcctcgagaa gatggatgga accgaggaac tcctcgtgaa gctcaataga 5521 gaggatcttc tcagaaagca gaggaccttc gataacggat ctatccctca tcagatccac 5581 ctcggagagt tgcacgctat ccttagaagg caagaggatt tctacccatt cctcaaggat 5641 aacagggaaa agattgagaa gattctcacc ttcagaatcc cttactacgt gggacctctc 5701 gctagaggaa actcaagatt cgcttggatg accagaaagt ctgaggaaac catcacccct 5761 tggaacttcg aagaggtggt ggataagggt gctagtgctc agtctttcat cgagaggatg 5821 accaacttcg ataagaacct tccaaacgag aaggtgctcc ctaagcactc tttgctctac 5881 gagtacttca ccgtgtacaa cgagttgacc aaggttaagt acgtgaccga gggaatgagg 5941 aagcctgctt ttttgtcagg tgagcaaaag aaggctatcg ttgatctctt gttcaagacc 6001 aacagaaagg tgaccgtgaa gcagctcaaa gaggattact tcaagaaaat cgagtgcttc 6061 gattcagttg agatttctgg tgttgaggat aggttcaacg catctctcgg aacctaccac 6121 gatctcctca agatcattaa ggataaggat ttcttggata acgaggaaaa cgaggatatc 6181 ttggaggata tcgttcttac cctcaccctc tttgaagata gagagatgat tgaagaaagg 6241 ctcaagacct acgctcatct cttcgatgat aaggtgatga agcagttgaa gagaagaaga 6301 tacactggtt ggggaaggct ctcaagaaag ctcattaacg gaatcaggga taagcagtct 6361 ggaaagacaa tccttgattt cctcaagt //