LOCUS Exported 6028 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial nuclease-null ST1 Cas9 expression. ACCESSION . VERSION . KEYWORDS DS-ST1casN- SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6028) AUTHORS Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM TITLE Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. JOURNAL Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681. PUBMED 24076762 REFERENCE 2 (bases 1 to 6028) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6028 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 153..180 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" CDS 377..3739 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus thermophilus Type II CRISPR/Cas system" /label=St1dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the mutations D9A, D598A, H599A, and N622A" /translation="MSDLVLGLAIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLV RRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEE LFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYG QLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGK RKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLL NDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRID KSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADG SFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKT TSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNED DEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERC LYTGKTISIHDLINNSNQFEVAAILPLSITFDDSLANKVLVYATAAQEKGQRTPYQALD SMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVV LNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNL WKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSY QVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSK FLMYRHDPQTFEKVIEPILENYPNKQINDKGKEVPCNPFLKYKEEHGYIRKYSKKGNGP EIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQ FDKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRT MPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQ HIIKNEGDKPKLDF" repeat_region 4067..4102 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" rep_origin 4284..5022 /direction=RIGHT /label=CloDF13 ori /note="Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins." promoter 5070..5161 /gene="bla" /label=AmpR promoter CDS 5162..5953 /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" ORIGIN 1 accaggcacg cctaaccgtc agtgagattg gatgagtgaa cgatattgat cgagaagagc 61 cctgcgcagc cgctgccgtg cctgcaggaa gcaacggccc ggagggtggc gggcaggacg 121 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt 181 tgcgtttcta caaactctgc tagcttctag agcacagcta acaccacgtc gtccctatct 241 gctgccctag gtctatgagt ggttgctgga taactttacg ggcatgcata aggctcgtat 301 gatatattca gggagaccac aacggtttcc ctctacaaat aattttgttt aacttttact 361 agaggaggag gcaaaaatga gcgacctggt gctgggcctg gccatcggca tcggcagcgt 421 gggcgtgggc atcctgaaca aggtgaccgg cgagatcatc cacaagaaca gtcgcatctt 481 ccctgctgct caggctgaga acaacctggt gcgccgcacc aaccgccagg gtcgccggct 541 tgctcgccgc aagaagcacc ggcgcgtgcg cctgaaccgc ctgttcgagg agagcggcct 601 gatcaccgac ttcaccaaga tcagcatcaa cctgaacccc taccagctgc gcgtgaaggg 661 cctgaccgac gagctgagca acgaggagct gttcatcgcc ctgaagaaca tggtgaagca 721 ccgcggcatc agctacctgg acgacgccag cgacgacggc aacagcagcg tgggcgacta 781 cgcccagatc gtgaaggaga acagcaagca gctggagacc aagacccccg gccagatcca 841 gctggagcgc taccagacct acggccagct gcgcggcgac ttcaccgtgg agaaggacgg 901 caagaagcac cgcctgatca acgtgttccc caccagcgcc taccgcagcg aggccctgcg 961 catcctgcag acccagcagg agttcaaccc ccagatcacc gacgagttca tcaaccgcta 1021 cctggagatc ctgaccggca agcgcaagta ctaccacggc cccggcaacg agaagagccg 1081 caccgactac ggccgctacc gcaccagcgg cgagaccctg gacaacatct tcggcatcct 1141 gatcggcaag tgcaccttct accccgacga gttccgcgcc gccaaggcca gctacaccgc 1201 ccaggagttc aacctgctga acgacctgaa caacctgacc gtgcccaccg agaccaagaa 1261 gctgagcaag gagcagaaga accagatcat caactacgtg aagaacgaga aggccatggg 1321 ccccgccaag ctgttcaagt acatcgccaa gctgctgagc tgcgacgtgg ccgacatcaa 1381 gggctaccgc atcgacaaga gcggcaaggc cgagatccac accttcgagg cctaccgcaa 1441 gatgaagacc ctggagaccc tggacatcga gcagatggac cgcgagaccc tggacaagct 1501 ggcctacgtg ctgaccctga acaccgagcg cgagggcatc caggaggccc tggagcacga 1561 gttcgccgac ggcagcttca gccagaagca ggtggacgag ctggtgcagt tccgcaaggc 1621 caacagcagc atcttcggca agggctggca caacttcagc gtgaagctga tgatggagct 1681 gatccccgag ctgtacgaga ccagcgagga gcagatgacc atcctgaccc gcctgggcaa 1741 gcagaagacc accagcagca gcaacaagac caagtacatc gacgagaagc tgctgaccga 1801 ggagatctac aaccccgtgg tggccaagag cgtgcgccag gccatcaaga tcgtgaacgc 1861 cgccatcaag gagtacggcg acttcgacaa catcgtgatc gagatggccc gcgagaccaa 1921 cgaggacgac gagaagaagg ccatccagaa gatccagaag gccaacaagg acgagaagga 1981 cgccgccatg ctgaaggccg ccaaccagta caacggcaag gccgagctgc cccacagcgt 2041 gttccacggc cacaagcagc tggccaccaa gatccgcctg tggcaccagc agggcgagcg 2101 ctgcctgtac accggcaaga ccatcagcat ccacgacctg atcaacaaca gcaaccagtt 2161 cgaggtggct gccatcctgc ccctgagcat caccttcgac gacagcctgg ccaacaaggt 2221 gctggtgtac gccaccgccg ctcaggagaa gggccagcgc accccctacc aggccctgga 2281 cagcatggac gacgcctgga gcttccgcga gctgaaggcc ttcgtgcgcg agagcaagac 2341 cctgagcaac aagaagaagg agtacctgct gaccgaggag gacatcagca agttcgacgt 2401 gcgcaagaag ttcatcgagc gcaacctggt ggacacccgc tacgccagcc gcgtggtgct 2461 gaacgccctg caggagcact tccgcgccca caagatcgac accaaggtga gcgtggtgcg 2521 cggccagttc accagccagc tgcgccgcca ctggggcatc gagaagaccc gcgacaccta 2581 ccaccaccac gccgtggacg ccctgatcat tgcggcttct agccagctga acctgtggaa 2641 gaagcagaag aacaccctgg tgagctacag cgaggaccag ctgctggaca tcgagaccgg 2701 cgagctgatc agcgacgacg agtacaagga gagcgtgttc aaggccccct accagcactt 2761 cgtggacacc ctgaagagca aggagttcga ggacagcatc ctgttcagct accaggtgga 2821 cagcaagttc aaccgcaaga tcagcgacgc caccatctac gccacccgcc aggccaaggt 2881 gggcaaggac aaggccgacg agacctacgt gctgggcaag atcaaggaca tctacaccca 2941 ggacggctac gacgccttca tgaagatcta caagaaggac aagagcaagt tcctgatgta 3001 ccgccacgac ccccagacct tcgagaaggt gatcgagccc atcctggaga actaccccaa 3061 caagcagatc aacgataaag gcaaggaggt gccctgcaac cccttcctga agtacaagga 3121 ggagcacggc tacatccgca agtacagcaa gaagggcaac ggccccgaga tcaagagcct 3181 gaagtactac gacagcaagc tgggcaacca catcgacatc acccccaagg acagcaacaa 3241 caaggtggtg ctgcagagcg tgagcccctg gcgcgccgac gtgtacttca acaagaccac 3301 cggcaagtac gagatcctgg gcctgaagta cgccgacctg cagtttgata agggcaccgg 3361 cacctacaag atcagccagg agaagtacaa cgacatcaag aagaaggagg gcgtggacag 3421 cgacagcgag ttcaagttca ccctgtacaa gaacgacctt ctgctggtga aggacaccga 3481 gaccaaggag caacagctgt tccgcttcct gagccgcacc atgcccaagc agaagcacta 3541 cgtggagctg aagccctacg acaagcagaa gttcgagggc ggcgaggccc tgatcaaggt 3601 gctgggcaac gtggccaaca gcggccagtg caagaagggc ctgggcaaga gcaacatcag 3661 catctacaag gtgcgcaccg acgtgctggg caaccagcac atcatcaaga acgagggcga 3721 caagcccaag ctggacttct aacatccgcg gcaccgccta agctagattt ttaatattaa 3781 ttgttagaaa gtgttgcaat tatagttatc atatactata ataatagtgt aagggacgcc 3841 ttacacagtt acttaaatct tgcagaagct acaaagataa ggcttcatgc cgaaatcaac 3901 accctgtcat tttatggcag ggtgttttcg ttatttaaag aggagaagaa atgacttgga 3961 gagttgcctg caggctccca tcttgggaca atgaaaacgt tagtcatggc gcgccttgac 4021 ggctagctca gtcctaggta cagtgctagc ttaattagtc tacgaggttt tagagctatg 4081 ctgttttgaa tggtcccaaa acctgcagac aagcccggcc ggcctaaggc gatgccccct 4141 cgacctcgat cagggaggcg ttcaggacga ctcacaaaga aagccgggca atgcccggct 4201 ttttccacgc ctcctgggct gacttcaggt gctacatttg aagagataaa ttgcactgaa 4261 atctagagcg gttcagtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 4321 gtaatctttt gccctgtaaa cgaaaaaacc acctggggag gtggtttgat cgaaggttaa 4381 gtcagttggg gaactgctta accgtggtaa ctggctttcg cagagcacag caaccaaatc 4441 tgtccttcca gtgtagccgg actttggcgc acacttcaag agcaaccgcg tgtttagcta 4501 aacaaatcct ctgcgaactc ccagttacca atggctgctg ccagtggcgt tttaccgtgc 4561 ttttccgggt tggactcaag tgaacagtta ccggataagg cgcagcagtc gggctgaacg 4621 gggagttctt gcttacagcc cagcttggag cgaacgacct acaccgagcc gagataccag 4681 tgtgtgagct atgagaaagc gccacacttc ccgtaaggga gaaaggcgga acaggtatcc 4741 ggtaaacggc agggtcggaa caggagagcg caagagggag cgacccgccg gaaacggtgg 4801 ggatctttaa gtcctgtcgg gtttcgcccg tactgtcaga ttcatggttg agcctcacgg 4861 ctcccacaga tgcaccggaa aagcgtctgt ttatgtgaac tctggcagga gggcggagcc 4921 tatggaaaaa cgccaccggc gcggccctgc tgttttgcct cacatgttag tcccctgctt 4981 atccacggaa tctgtgggta actttgtatg tgtccgcagc gcccgccgca gtctcacgcc 5041 cggagcgtag cgaccgagtg agctagctat ttgtttattt ttctaaatac attcaaatat 5101 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 5161 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat 5221 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg 5281 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga 5341 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag 5401 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg 5461 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg 5521 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga 5581 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca 5641 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc 5701 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg 5761 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta 5821 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc 5881 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt 5941 agtcggcaaa taatgtctaa caattcgttc aagccgaggg gccgcaagat ccggccacga 6001 tgacccggtc gtcggttcag ggcagggt //