LOCUS Exported 6794 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION dCas9 expression unit plasmid. The dCas9 expression unit plasmids contain connector ConL1, ConRE, and one dCas9 transcriptional unit. . ACCESSION . VERSION . KEYWORDS TDK promoter dCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6794) AUTHORS Geng P, Leonard SP, Mishler DM, Barrick JE TITLE Synthetic Genome Defenses against Selfish DNA Elements Stabilize Engineered Bacteria against Evolutionary Failure. JOURNAL ACS Synth Biol. 2019 Mar 15;8(3):521-531. doi: 10.1021/acssynbio.8b00426. Epub 2019 Feb 15. PUBMED 30703321 REFERENCE 2 (bases 1 to 6794) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6794 /organism="synthetic DNA construct" /mol_type="other DNA" RBS 238..249 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 258..4364 /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 NLIGALLFDSGETAEATRHKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" misc_feature 4385..4516 /label=crRNA leader /note="crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 4517..4552 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" terminator 4568..4616 /label=fd terminator /note="central terminator from bacteriophage fd (Otsuka and Kunisawa, 1982)" CDS complement(5027..5887) /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 5650..5669 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5888..5992) /gene="bla" /label=AmpR promoter rep_origin 6095..6683 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6584..6603 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gaattcgcat ctagaacggg gtcatcacgg ctcatcatgc gccaaacaaa tgtgtgcaat 61 acacgctcgg atgactgcat gatgaccgca ctgactgggg acagcagatc cacctaagcc 121 tgtgagagaa gcagacaccc gacagatcaa ggcagttacg tctcaccaaa ccagatgtca 181 acacagctac aacgcccagc ctccaattca aatcataaaa aatttatttg ctttattaaa 241 gaggagaaat taattatatg gataagaaat actcaatagg cttagctatc ggcacaaata 301 gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc 361 tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca 421 gtggagagac agcggaagcg actcgtcata aacggacagc tcgtagaagg tatacacgtc 481 ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg 541 atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac 601 gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta 661 tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct 721 atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa 781 atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat 841 tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac 901 gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa 961 atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa 1021 attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt 1081 tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga 1141 atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg 1201 ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt 1261 taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat 1321 caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat 1381 ttatcaaacc aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc 1441 gtgaagattt gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc 1501 acttgggtga gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag 1561 acaatcgtga gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat 1621 tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc 1681 catggaattt tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca 1741 tgacaaactt tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt 1801 atgagtattt tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc 1861 gaaaaccagc atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa 1921 caaatcgaaa agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt 1981 ttgatagtgt tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc 2041 atgatttgct aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata 2101 tcttagagga tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa 2161 gacttaaaac atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc 2221 gttatactgg ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat 2281 ctggcaaaac aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc 2341 agctgatcca tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg 2401 gacaaggcga tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa 2461 aaggtatttt acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata 2521 agccagaaaa tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga 2581 aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga 2641 ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt 2701 atctccaaaa tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg 2761 attatgatgt cgatgccatt gttccacaaa gtttccttaa agacgattca atagacaata 2821 aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag 2881 tagtcaaaaa gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac 2941 gtaagtttga taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg 3001 gttttatcaa acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt 3061 tggatagtcg catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag 3121 tgattacctt aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag 3181 tacgtgagat taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa 3241 ctgctttgat taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag 3301 tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa 3361 aatatttctt ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg 3421 gagagattcg caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg 3481 ataaagggcg agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg 3541 tcaagaaaac agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa 3601 attcggacaa gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg 3661 atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga 3721 agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg 3781 aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa 3841 tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg 3901 ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt 3961 ttttatattt agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa 4021 aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg 4081 aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata 4141 acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt 4201 tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac 4261 gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc 4321 tttatgaaac acgcattgat ttgagtcagc taggaggtga ctgaagtata ttttagatga 4381 agattatttc ttaataacta aaaatatggt ataatactct taataaatgc agtaatacag 4441 gggcttttca agactgaagt ctagctgaga caaatagtgc gattacgaaa ttttttagac 4501 aaaaatagtc tacgaggttt tagagctatg ctgttttgaa tggtcccaaa acaaagcaag 4561 ctgataaacc gatacaatta aaggctcctt ttggagcctt tttttttgga gattttcaac 4621 atgaagaaat tattatttga tgatcagata gcggcgggga actgccagac atcaaataaa 4681 acaaaaggct cagtcggaag actgggcctt ttgttttatc tgttgtttgt cggtgaacac 4741 tctcccggct gagcatgaga cggaaatctg ctcgtcagtg gtgctcacac tgacgaatca 4801 tgtacagatc ataccgatga ctgcctggcg actcacaact aagcaagaca gccggaacca 4861 gcgccggcga acaccactgc atatatggca tatcacaaca gtccaactag tgcactgcag 4921 tacagcggcc gcgattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4981 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5041 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 5101 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 5161 taccgcggga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 5221 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 5281 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 5341 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 5401 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 5461 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 5521 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 5581 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 5641 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 5701 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 5761 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 5821 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 5881 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 5941 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 6001 cccgaaaagt gccacctgtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 6061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 6121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 6181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 6241 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 6301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 6361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 6421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 6481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 6541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 6601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 6661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 6721 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 6781 gtgcggccgc ccct //