LOCUS Exported 9706 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION dCas9 plasmid expressing 3A sRNA trigger. ACCESSION . VERSION . KEYWORDS PAJ390 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9706) AUTHORS Galizi R, Duncan JN, Rostain W, Quinn CM, Storch M, Kushwaha M, Jaramillo A TITLE Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics. JOURNAL CRISPR J. 2020 Oct;3(5):398-408. doi: 10.1089/crispr.2020.0029. PUBMED 33095053 REFERENCE 2 (bases 1 to 9706) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9706 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(1708..2367) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2282..2301 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(2368..2470) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" protein_bind 2855..2873 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2880..2898 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind complement(3242..3259) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3376..3921) /direction=LEFT /label=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. " primer_bind complement(3456..3475) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 4033..4061 /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene" misc_RNA complement(4379..4457) /label=tracrRNA /note="trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 4753..8859 /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" misc_feature 8880..9011 /label=crRNA leader /note="crRNA leader sequence for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 9012..9047 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" repeat_region 9078..9113 /label=DR /note="direct repeat for the Streptococcus pyogenes CRISPR/Cas system" primer_bind complement(9542..9561) /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 acctactact gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgaccgatgc 61 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg 121 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 181 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg 241 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac 301 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg ggctacgtct 361 tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt ctcgcttccg 421 gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat gacgaccatc 481 agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc attggaccgc 541 tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg gcatggattg 601 taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg 661 ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat 721 tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat 781 atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc 841 ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg 901 ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg 961 ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta 1021 aagtctggaa acgcggaagt cccctacgtg ctgctgaagt tgcccgcaac agagagtgga 1081 accaaccggt gataccacga tactatgact gagagtcaac gccatgagcg gcctcatttc 1141 ttattctgag ttacaacagt ccgcaccgct gccggtagct ccttccggtg ggcgcggggc 1201 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg 1261 ccggcagcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa 1321 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga 1381 acacctacat ctgtattaac gaagcgctaa ccgtttttat caggctctgg gaggcagaat 1441 aaatgatcat atcgtcaatt attacctcca cggggagagc ctgagcaaac tggcctcagg 1501 catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat 1561 agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt 1621 cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcaac caggcgttta 1681 agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagtactg 1741 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg 1801 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac 1861 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca 1921 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt 1981 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg 2041 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg 2101 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg 2161 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt 2221 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg 2281 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt 2341 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa 2401 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac 2461 gtgccgatca acgtctcatt ttcgccaaaa gttggcccgg ggcttcccgg tatcaacagg 2521 gacaccagga tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca 2581 aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt ttttgaggtg 2641 ctccagtggc ttctgtttct atcagctgtc cctcctgttc agctactgac agcttgattc 2701 tagctgatcg tggaccggaa ggtgagccag tgagttgatt gcagtccagt tacgctggag 2761 tctgaggctc gtcctgaatg atatgcgacc gccggagggt tgcgtttgag acgggcgaca 2821 gatcgacact gctcgatccg ctcgcaccga attctcccta tcagtgatag agattgacat 2881 ccctatcagt gatagagata ctgagcacgg cagacaaaga aacctacgtt gaacagcatg 2941 aagtcgcatc cttagcgaaa gctaaggatt ttttttctgc agggatcgac gagagcagcg 3001 cgactggatc agttctggac gagcgagctg tcgtccgacc cgtgatctta cggcattata 3061 cgtatgatcg ggggtggtgc gtaacggcaa aagcaccgcc ggacatcagc gctagcggag 3121 tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca 3181 ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt 3241 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg 3301 aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc 3361 cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg 3421 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3481 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt 3541 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca 3601 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact 3661 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta 3721 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca 3781 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag 3841 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg 3901 cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt 3961 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg 4021 taattctaat gtttgacagc ttatcatcga taagctttaa tgcggtagtt tatcacagtt 4081 aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca tcgtcatcct 4141 cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct 4201 cttgcgggat tacgaaatca tcctgtggag cttagtaggt ttagcaagat ggcagcgcct 4261 aaatgtagaa tgataaaagg attaagagat taatttccct aaaaatgata aaacaagcgt 4321 tttgaaagcg cttgtttttt tggtttgcag tcagagtaga atagaagtat caaaaaaagc 4381 accgactcgg tgccactttt tcaagttgat aacggactag ccttatttta acttgctatg 4441 ctgttttgaa tggttccaac aagattattt tataactttt ataacaaata atcaaggaga 4501 aattcaaaga aatttatcag ccataaaaca atacttaata ctatagaatg ataacaaaat 4561 aaactacttt ttaaaagaat tttgtgttat aatctattta ttattaagta ttgggtaata 4621 ttttttgaag agatattttg aaaaagaaaa attaaagcat attaaactaa tttcggaggt 4681 cattaaaact attattgaaa tcatcaaact cattatggat ttaatttaaa ctttttattt 4741 taggaggcaa aaatggataa gaaatactca ataggcttag ctatcggcac aaatagcgtc 4801 ggatgggcgg tgatcactga tgaatataag gttccgtcta aaaagttcaa ggttctggga 4861 aatacagacc gccacagtat caaaaaaaat cttatagggg ctcttttatt tgacagtgga 4921 gagacagcgg aagcgactcg tctcaaacgg acagctcgta gaaggtatac acgtcggaag 4981 aatcgtattt gttatctaca ggagattttt tcaaatgaga tggcgaaagt agatgatagt 5041 ttctttcatc gacttgaaga gtcttttttg gtggaagaag acaagaagca tgaacgtcat 5101 cctatttttg gaaatatagt agatgaagtt gcttatcatg agaaatatcc aactatctat 5161 catctgcgaa aaaaattggt agattctact gataaagcgg atttgcgctt aatctatttg 5221 gccttagcgc atatgattaa gtttcgtggt cattttttga ttgagggaga tttaaatcct 5281 gataatagtg atgtggacaa actatttatc cagttggtac aaacctacaa tcaattattt 5341 gaagaaaacc ctattaacgc aagtggagta gatgctaaag cgattctttc tgcacgattg 5401 agtaaatcaa gacgattaga aaatctcatt gctcagctcc ccggtgagaa gaaaaatggc 5461 ttatttggga atctcattgc tttgtcattg ggtttgaccc ctaattttaa atcaaatttt 5521 gatttggcag aagatgctaa attacagctt tcaaaagata cttacgatga tgatttagat 5581 aatttattgg cgcaaattgg agatcaatat gctgatttgt ttttggcagc taagaattta 5641 tcagatgcta ttttactttc agatatccta agagtaaata ctgaaataac taaggctccc 5701 ctatcagctt caatgattaa acgctacgat gaacatcatc aagacttgac tcttttaaaa 5761 gctttagttc gacaacaact tccagaaaag tataaagaaa tcttttttga tcaatcaaaa 5821 aacggatatg caggttatat tgatggggga gctagccaag aagaatttta taaatttatc 5881 aaaccaattt tagaaaaaat ggatggtact gaggaattat tggtgaaact aaatcgtgaa 5941 gatttgctgc gcaagcaacg gacctttgac aacggctcta ttccccatca aattcacttg 6001 ggtgagctgc atgctatttt gagaagacaa gaagactttt atccattttt aaaagacaat 6061 cgtgagaaga ttgaaaaaat cttgactttt cgaattcctt attatgttgg tccattggcg 6121 cgtggcaata gtcgttttgc atggatgact cggaagtctg aagaaacaat taccccatgg 6181 aattttgaag aagttgtcga taaaggtgct tcagctcaat catttattga acgcatgaca 6241 aactttgata aaaatcttcc aaatgaaaaa gtactaccaa aacatagttt gctttatgag 6301 tattttacgg tttataacga attgacaaag gtcaaatatg ttactgaagg aatgcgaaaa 6361 ccagcatttc tttcaggtga acagaagaaa gccattgttg atttactctt caaaacaaat 6421 cgaaaagtaa ccgttaagca attaaaagaa gattatttca aaaaaataga atgttttgat 6481 agtgttgaaa tttcaggagt tgaagataga tttaatgctt cattaggtac ctaccatgat 6541 ttgctaaaaa ttattaaaga taaagatttt ttggataatg aagaaaatga agatatctta 6601 gaggatattg ttttaacatt gaccttattt gaagataggg agatgattga ggaaagactt 6661 aaaacatatg ctcacctctt tgatgataag gtgatgaaac agcttaaacg tcgccgttat 6721 actggttggg gacgtttgtc tcgaaaattg attaatggta ttagggataa gcaatctggc 6781 aaaacaatat tagatttttt gaaatcagat ggttttgcca atcgcaattt tatgcagctg 6841 atccatgatg atagtttgac atttaaagaa gacattcaaa aagcacaagt gtctggacaa 6901 ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt 6961 attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca 7021 gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat 7081 tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt 7141 aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc 7201 caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat 7261 gatgtcgatg ccattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc 7321 ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc 7381 aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag 7441 tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt 7501 atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat 7561 agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt 7621 accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt 7681 gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct 7741 ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat 7801 gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat 7861 ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag 7921 attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa 7981 gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag 8041 aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg 8101 gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt 8161 ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag 8221 ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa 8281 aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt 8341 aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt 8401 gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta 8461 tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa 8521 ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt 8581 tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa 8641 catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg 8701 aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat 8761 acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat 8821 gaaacacgca ttgatttgag tcagctagga ggtgactgaa gtatatttta gatgaagatt 8881 atttcttaat aactaaaaat atggtataat actcttaata aatgcagtaa tacaggggct 8941 tttcaagact gaagtctagc tgagacaaat agtgcgatta cgaaattttt tagacaaaaa 9001 tagtctacga ggttttagag ctatgctgtt ttgaatggtc ccaaaactga gaccagtctc 9061 ggaagctcaa aggtctcgtt ttagagctat gctgttttga atggtcccaa aacttcagca 9121 cactgagact tgttgagttc catgttttag agctatgctg ttttgaatgg actccattca 9181 acattgccga tgataacttg agaaagaggg ttaataccag cagtcggata ccttcctatt 9241 ctttctgtta aagcgttttc atgttataat aggcaaaaga agagtagtgt gatcgtccat 9301 tccgacagca tcgccagtca ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt 9361 ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg gccgccgccc agtcctgctc 9421 gcttcgctac ttggagccac tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg 9481 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc 9541 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc 9601 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc 9661 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctca //