LOCUS Exported 9764 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expression of dCas9 driven by lux cassette from Vibrio fischeri. ACCESSION . VERSION . KEYWORDS pQdCas9-sgempty SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9764) AUTHORS Liu Y, Chen J, Crisante D, Jaramillo Lopez JM, Mahadevan R TITLE Dynamic Cell Programming with Quorum Sensing-Controlled CRISPRi Circuit. JOURNAL ACS Synth Biol. 2020 Jun 5. doi: 10.1021/acssynbio.0c00148. PUBMED 32485106 REFERENCE 2 (bases 1 to 9764) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9764 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..858 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(118..137) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 728..747 /label=Neo-F /note="Neomycin resistance gene, forward primer" RBS 2596..2607 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 2614..6720 /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" promoter 6727..6761 /label=J23119(SpeI) promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119) modified to end with an SpeI site" misc_RNA 6762..6837 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS complement(6916..7578) /codon_start=1 /product="replication protein for the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica" /label=pBBR1 Rep /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR" rep_origin 7579..8348 /label=pBBR1 oriV /note="replication origin of the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica; requires the pBBR1 Rep protein for replication" ORIGIN 1 ccaaggatct gatggcgcag gggatcaaga tttgatcaag agacaggatg aggatcgttt 61 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta 121 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg 181 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa 241 ctacaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct 301 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg 361 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca 421 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat 481 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac 541 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc 601 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa 661 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag 721 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 781 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt 841 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca 901 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg gactagagaa 961 ttcatcctga acttatctag attaatttaa gactgctttt ttaaactgtt cattaatagg 1021 catagacaat acaaccgatt tagtatcacc taatacatga atttctttgt ctccaatacg 1081 atgacaagga actttaatac gctttaaaaa tcgctctatt gctgttgatg ttactgttac 1141 atattctgta ataccttgac taacagcgtg tttatatata gcttcaaata gtttcattgt 1201 aatttcacta gcagagttat ttatctttga gctattttta cctacagcaa aacgacttaa 1261 ttcgactata ttaggatctt tgggagcact ctgttgacca agcaattcag gaaaaacact 1321 tttcagcata taatcacctg ttgtaggtaa taaacgccag catccactta cattttcagt 1381 atcatcacaa gcataaatat attctgcatt tgagttatca tactcatctg attcaaggtt 1441 attttctaca actaagtccc actcaagtct ttgcttaaac acttgataac gaagacttag 1501 aataccttta tactcctccg atggaattgc caaaaaatcc gattttttta tcattatagt 1561 cataccaacc tcccttgcgt ttattcgact ataacaaacc attttcttgc gtaaacctgt 1621 acgatcctac aggtgcttat gttaagtaat tgtattccca gcgatacaat agtgtgacaa 1681 aaatccaatt tattagaatc aaatgtcaat ccattaccgt tttaatgata tataacacgc 1741 aaaacttgcg acaaacaata ggtaaggata aagagatggg tatgaaaaac ataaatgccg 1801 acgacacata cagaataatt aataaaatta aagcttgtag aagcaataat gatattaatc 1861 aatgcttatc tgatatgact aaaatggtac attgtgaata ttatttactc gcgatcattt 1921 atcctcattc tatggttaaa tctgatattt caattctaga taattaccct aaaaaatgga 1981 ggcaatatta tgatgacgct aatttaataa aatatgatcc tatagtagat tattctaact 2041 ccaatcattc accaattaat tggaatatat ttgaaaacaa tgctgtaaat aaaaaatctc 2101 caaatgtaat taaagaagcg aaaacatcag gtcttatcac tgggtttagt ttccctattc 2161 atacggctaa caatggcttc ggaatgctta gttttgcaca ttcagaaaaa gacaactata 2221 tagatagttt atttttacat gcgtgtatga acataccatt aattgttcct tctctagttg 2281 ataattatcg aaaaataaat atagcaaata ataaatcaaa caacgattta accaaaagag 2341 aaaaagaatg tttagcgtgg gcatgcgaag gaaaaagctc ttgggatatt tcaaaaatat 2401 taggctgcag tgagcgtact gtcactttcc atttaaccaa tgcgcaaatg aaactcaata 2461 caacaaaccg ctgccaaagt atttctaaag caattttaac aggagcaatt gattgcccat 2521 actttaaaaa ttaaagatct acctgtagga tcgtacaggt ttacgcaaga aaatggtttg 2581 ttatagtcga ataaaaaaga ggagaaagga tctatggata agaaatactc aataggctta 2641 gctatcggca caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct 2701 aaaaagttca aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg 2761 gctcttttat ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt 2821 agaaggtata cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag 2881 atggcgaaag tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa 2941 gacaagaagc atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat 3001 gagaaatatc caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg 3061 gatttgcgct taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg 3121 attgagggag atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta 3181 caaacctaca atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa 3241 gcgattcttt ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc 3301 cccggtgaga agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc 3361 cctaatttta aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat 3421 acttacgatg atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg 3481 tttttggcag ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat 3541 actgaaataa ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat 3601 caagacttga ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa 3661 atcttttttg atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa 3721 gaagaatttt ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta 3781 ttggtgaaac taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct 3841 attccccatc aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt 3901 tatccatttt taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct 3961 tattatgttg gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct 4021 gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa 4081 tcatttattg aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca 4141 aaacatagtt tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat 4201 gttactgaag gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt 4261 gatttactct tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc 4321 aaaaaaatag aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct 4381 tcattaggta cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat 4441 gaagaaaatg aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg 4501 gagatgattg aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa 4561 cagcttaaac gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt 4621 attagggata agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc 4681 aatcgcaatt ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa 4741 aaagcacaag tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt 4801 agccctgcta ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa 4861 gtaatggggc ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca 4921 actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa 4981 gaattaggaa gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa 5041 aagctctatc tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat 5101 attaatcgtt taagtgatta tgatgtcgat gccattgttc cacaaagttt ccttaaagac 5161 gattcaatag acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac 5221 gttccaagtg aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc 5281 aagttaatca ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt 5341 gaacttgata aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag 5401 catgtggcac aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt 5461 attcgagagg ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat 5521 ttccaattct ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta 5581 aatgccgtcg ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc 5641 tatggtgatt ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata 5701 ggcaaagcaa ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa 5761 attacacttg caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact 5821 ggagaaattg tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg 5881 ccccaagtca atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca 5941 attttaccaa aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa 6001 aaatatggtg gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg 6061 gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg 6121 gaaagaagtt cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa 6181 gttaaaaaag acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt 6241 cgtaaacgga tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca 6301 agcaaatatg tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca 6361 gaagataacg aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt 6421 attgagcaaa tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa 6481 gttcttagtg catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt 6541 attcatttat ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca 6601 acaattgatc gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat 6661 caatccatca ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactaa 6721 ctcgagttga cagctagctc agtcctaggt ataatactag tgttttagag ctagaaatag 6781 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 6841 ttttgttaac ttcaggacct gcagtctagt atagtctgga acagcgcact tacgggttgc 6901 tgcgcaaccc aagtgctacc ggcgcggcag cgtgacccgt gtcggcggct ccaacggctc 6961 gccatcgtcc agaaaacacg gctcatcggg catcggcagg cgctgctgcc cgcgccgttc 7021 ccattcctcc gtttcggtca aggctggcag gtctggttcc atgcccggaa tgccgggctg 7081 gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc tcgcccggat acagggtcgg 7141 gatgcggcgc aggtcgccat gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac 7201 cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg 7261 gtcattgacc acgtaggccg acacggtgcc ggggccgttg agcttcacga cggagatcca 7321 gcgctcggcc accaagtcct tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag 7381 cttggaaagt gtcttctggc tgaccaccac ggcgttctgg tggcccatct gcgccacgag 7441 gtgatgcagc agcattgccg ccgtgggttt cctcgcaata agcccggccc acgcctcatg 7501 cgctttgcgt tccgtttgca cccagtgacc gggcttgttc ttggcttgaa tgccgatttc 7561 tctggactgc gtggccatgc ttatctccat gcggtagggt gccgcacggt tgcggcacca 7621 tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg 7681 cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg 7741 agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct 7801 atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg 7861 cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc 7921 ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc 7981 tcggggggca ggcgggcggg cttcgccttc gactgccccc actcgcatag gcttgggtcg 8041 ttccaggcgc gtcaaggcca agccgctgcg cggtcgctgc gcgagccttg acccgccttc 8101 cacttggtgt ccaaccggca agcgaagcgc gcaggccgca ggccggaggc ttttccccag 8161 agaaaattaa aaaaattgat ggggcaaggc cgcaggccgc gcagttggag ccggtgggta 8221 tgtggtcgaa ggctgggtag ccggtgggca atccctgtgg tcaagctcgt gggcaggcgc 8281 agcctgtcca tcagcttgtc cagcagggtt gtccacgggc cgagcgaagc gagccagccg 8341 gtggccgctc gcggccatcg tccacatatc cacgggctgg caagggagcg cagcgaccgc 8401 gcagggcgaa gcccggagag caagcccgta gggcgccgca gccgccgtag gcggtcacga 8461 ctttgcgaag caaagtctag tgagtatact caagcattga gtggcccgcc ggaggcaccg 8521 ccttgcgctg cccccgtcga gccggttgga caccaaaagg gaggggcagg catggcggca 8581 tacgcgatca tgcgatgcaa gaagctggcg aaaatgggca acgtggcggc cagtctcaag 8641 cacgcctacc gcgagcgcga gacgcccaac gctgacgcca gcaggacgcc agagaacgag 8701 cactgggcgg ccagcagcac cgatgaagcg atgggccgac tgcgcgagtt gctgccagag 8761 aagcggcgca aggacgctgt gttggcggtc gagtacgtca tgacggccag cccggaatgg 8821 tggaagtcgg ccagccaaga acagcaggcg gcgttcttcg agaaggcgca caagtggctg 8881 gcggacaagt acggggcgga tcgcatcgtg acggccagca tccaccgtga cgaaaccagc 8941 ccgcacatga ccgcgttcgt ggtgccgctg acgcaggacg gcaggctgtc ggccaaggag 9001 ttcatcggca acaaagcgca gatgacccgc gaccagacca cgtttgcggc tgctgtggcc 9061 gatctagggc tgcaacgggg catcgagggc agcaaggcac gtcacacgcg cattcaggcg 9121 ttctacgagg ccctggagcg gccaccagtg ggccacgtca ccatcagccc gcaagcggtc 9181 gagccacgcg cctatgcacc gcagggattg gccgaaaagc tgggaatctc aaagcgcgtt 9241 gagacgccgg aagccgtggc cgaccggctg acaaaagcgg ttcggcaggg gtatgagcct 9301 gccctacagg ccgccgcagg agcgcgtgag atgcgcaaga aggccgatca agcccaagag 9361 acggcccgag accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat 9421 cgggatatgc aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg 9481 gaacagcggg aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca 9541 catttccccg aaaagtgcca cctgggatga atgtcagcta ctgggctatc tggacaaggg 9601 aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag 9661 actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta 9721 aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccg //