LOCUS Exported 8073 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION dxCas9(3.7), MCP-SoxS(R93A/S101A), J306 scRNA. ACCESSION . VERSION . KEYWORDS pCD565 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8073) AUTHORS Fontana J, Dong C, Kiattisewee C, Chavali VP, Tickman BI, Carothers JM, Zalatan JG TITLE Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements. JOURNAL Nat Commun. 2020 Apr 1;11(1):1618. doi: 10.1038/s41467-020-15454-y. PUBMED 32238808 REFERENCE 2 (bases 1 to 8073) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8073 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA complement(209..287) /label=tracrRNA /note="trans-activating CRISPR RNA for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 583..4686 /codon_start=1 /product="catalytically dead Streptococcus pyogenes Cas9 mutant with broad PAM compatibility (Hu et al., 2018)" /label=dxCas9(3.7) /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" terminator 4713..4784 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4800..4827 /label=T7Te terminator /note="phage T7 early transcription terminator" RBS 4924..4935 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" rep_origin complement(5837..6382) /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(5917..5936) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 6490..6524 /label=J23119(SpeI) promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119) modified to end with an SpeI site" misc_RNA 6608..6626 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" CDS 6649..6678 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 6694..6711 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(6767..6784) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 6937..6983 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 7201..7295 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(7316..7975) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 7890..7909 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(7976..5) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" ORIGIN 1 gatcaacgtc tcattttcgc cagatatcga cgtcttaagt tacgaaatca tcctgtggag 61 cttagtaggt ttagcaagat ggcagcgcct aaatgtagaa tgataaaagg attaagagat 121 taatttccct aaaaatgata aaacaagcgt tttgaaagcg cttgtttttt tggtttgcag 181 tcagagtaga atagaagtat caaaaaaagc accgactcgg tgccactttt tcaagttgat 241 aacggactag ccttatttta acttgctatg ctgttttgaa tggttccaac aagattattt 301 tataactttt ataacaaata atcaaggaga aattcaaaga aatttatcag ccataaaaca 361 atacttaata ctatagaatg ataacaaaat aaactacttt ttaaaagaat tttgtgttat 421 aatctattta ttattaagta ttgggtaata ttttttgaag agatattttg aaaaagaaaa 481 attaaagcat attaaactaa tttcggaggt cattaaaact attattgaaa tcatcaaact 541 cattatggat ttaatttaaa ctttttattt taggaggcaa aaatggacaa gaagtactcc 601 attgggctcg ctatcggcac aaacagcgtc ggctgggccg tcattacgga cgagtacaag 661 gtgccgagca aaaaattcaa agttctgggc aataccgatc gccacagcat aaagaagaac 721 ctcattggcg ccctcctgtt cgactccggg gagacggccg aagccacgcg gctcaaaaga 781 acagcacggc gcagatatac ccgcagaaag aatcggatct gctacctgca ggagatcttt 841 agtaatgaga tggctaaggt ggatgactct ttcttccata ggctggagga gtcctttttg 901 gtggaggagg ataaaaagca cgagcgccac ccaatctttg gcaatatcgt ggacgaggtg 961 gcgtaccatg aaaagtaccc aaccatatat catctgagga agaagcttgt agacagtact 1021 gataaggctg acttgcggtt gatctatctc gcgctggcgc atatgatcaa atttcgggga 1081 cacttcctca tcgaggggga cctgaaccca gacaacagcg atgtcgacaa actctttatc 1141 caactggttc agacttacaa tcagcttttc gaagagaacc cgatcaacgc atccggagtt 1201 gacgccaaag caatcctgag cgctaggctg tccaaatccc ggcggctcga aaacctcatc 1261 gcacagctcc ctggggagaa gaagaacggc ctgtttggta atcttatcgc cctgtccctc 1321 gggctgaccc ccaactttaa atctaacttc gacctggccg aagataccaa gcttcaactg 1381 agcaaagaca cctacgatga tgatctcgac aatctgctgg cccagatcgg cgaccagtac 1441 gcagaccttt ttttggcggc aaagaacctg tcagacgcca ttctgctgag tgatattctg 1501 cgagtgaaca cggagatcac caaagctccg ctgagcgcta gtatgatcaa gctctatgat 1561 gagcaccacc aagacttgac tttgctgaag gcccttgtca gacagcaact gcctgagaag 1621 tacaaggaaa ttttcttcga tcagtctaaa aatggctacg ccggatacat tgacggcgga 1681 gcaagccagg aggaatttta caaatttatt aagcccatct tggaaaaaat ggacggcacc 1741 gaggagctgc tggtaaagct taacagagaa gatctgttgc gcaaacagcg cactttcgac 1801 aatggaatca tcccccacca gattcacctg ggcgaactgc acgctatcct caggcggcaa 1861 gaggatttct accccttttt gaaagataac agggaaaaga ttgagaaaat cctcacattt 1921 cggataccct actatgtagg ccccctcgcc cggggaaatt ccagattcgc gtggatgact 1981 cgcaaatcag aagagaccat cactccctgg aacttcgaga aagtcgtgga taagggggcc 2041 tctgcccagt ccttcatcga aaggatgact aactttgata aaaatctgcc taacgaaaag 2101 gtgcttccta aacactctct gctgtacgag tacttcacag tttataacga gctcaccaag 2161 gtcaaatacg tcacagaagg gatgagaaag ccagcattcc tgtctggaga tcagaagaaa 2221 gctattgtgg acctcctctt caagacgaac cggaaagtta ccgtgaaaca gctcaaagaa 2281 gactatttca aaaagattga atgtttcgac tctgttgaaa tcagcggagt ggaggatcgc 2341 ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa tcattaaaga caaggacttc 2401 ctggacaatg aggagaacga ggacattctt gaggacattg tcctcaccct tacgttgttt 2461 gaagataggg agatgattga agaacgcttg aaaacttacg ctcatctctt cgacgacaaa 2521 gtcatgaagc agctcaagag gcgccgatat acaggatggg ggcggctgtc aagaaaactg 2581 atcaatggga tccgagacaa gcagagtgga aagacaatcc tggattttct taagtccgat 2641 ggatttgcca accggaactt cattcagttg atccatgatg actctctcac ctttaaggag 2701 gacatccaga aagcacaagt ttctggccag ggggacagtc ttcacgagca catcgctaat 2761 cttgcaggta gcccagctat caaaaaggga atactgcaga ccgttaaggt cgtggatgaa 2821 ctcgtcaaag taatgggaag gcataagccc gagaatatcg ttatcgagat ggcccgagag 2881 aaccaaacca cccagaaggg acagaagaac agtagggaaa ggatgaagag gattgaagag 2941 ggtataaaag aactggggtc ccaaatcctt aaggaacacc cagttgaaaa cacccagctt 3001 cagaatgaga agctctacct gtactacctg cagaacggca gggacatgta cgtggatcag 3061 gaactggaca tcaatcggct ctccgactac gacgtggacg ctatcgtgcc ccagtctttt 3121 ctcaaagatg attctattga taataaagtg ttgacaagat ccgataaaaa cagagggaag 3181 agtgataacg tcccctcaga agaagttgtc aagaaaatga aaaattattg gcggcagctg 3241 ctgaacgcca aactgatcac acaacggaag ttcgataatc tgactaaggc tgaacgaggt 3301 ggcctgtctg agttggataa agccggtttc atcaaaaggc agcttgttga gacacgccag 3361 atcaccaagc acgtggccca aattctcgat tcacgcatga acaccaagta cgatgaaaat 3421 gacaaactga ttcgagaggt gaaagttatt actctgaagt ctaagctggt ctcagatttc 3481 agaaaggact ttcagtttta taaggtgaga gagatcaaca attaccacca tgcgcatgat 3541 gcctacctga atgcagtggt aggcactgca cttatcaaaa aatatcccaa gcttgaatct 3601 gaatttgttt acggagacta taaagtgtac gatgttagga aaatgatcgc aaagtctgag 3661 caggaaatag gcaaggccac cgctaagtac ttcttttaca gcaatattat gaattttttc 3721 aagaccgaga ttacactggc caatggagag attcggaagc gaccacttat cgaaacaaac 3781 ggagaaacag gagaaatcgt gtgggacaag ggtagggatt tcgcgacagt ccggaaggtc 3841 ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag tacagaccgg aggtttctcc 3901 aaggaaagta tcctcccgaa aaggaacagc gacaagctga tcgcacgcaa aaaagattgg 3961 gaccccaaga aatacggcgg attcgattct cctacagtcg cttacagtgt actggttgtg 4021 gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg tcaaggaact gctgggcatc 4081 acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg actttctcga ggcgaaagga 4141 tataaagagg tcaaaaaaga cctcatcatt aagcttccca agtactctct ctttgagctt 4201 gaaaacggcc ggaaacgaat gctcgctagt gcgggcgtgc tgcagaaagg taacgagctg 4261 gcactgccct ctaaatacgt taatttcttg tatctggcca gccactatga aaagctcaaa 4321 gggtctcccg aagataatga gcagaagcag ctgttcgtgg aacaacacaa acactacctt 4381 gatgagatca tcgagcaaat aagcgaattc tccaaaagag tgatcctcgc cgacgctaac 4441 ctcgataagg tgctttctgc ttacaataag cacagggata agcccatcag ggagcaggca 4501 gaaaacatta tccacttgtt tactctgacc aacttgggcg cgcctgcagc cttcaagtac 4561 ttcgacacta ccatagacag aaagcggtac acctctacaa aggaggtcct ggacgccaca 4621 ctgattcatc agtcaattac ggggctctat gaaacaagaa tcgacctctc tcagctcggt 4681 ggagactaac tcgagtaagg atctccaggc atcaaataaa acgaaaggct cagtcgaaag 4741 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg 4801 gctcaccttc gggtgggcct ttctgcgttt atacctagag cgcccggaag agagtcaatt 4861 cagggtggtg aatctgcagt ttacggctag ctcagcccta ggtattatgc tagcgaattc 4921 attaaagagg agaaaggtac catggggccc gcttctaact ttactcagtt cgttctcgtc 4981 gacaatggcg gaactggcga cgtgactgtc gccccaagca acttcgctaa cgggatcgct 5041 gaatggatca gctctaactc gcgttcacag gcttacaaag taacctgtag cgttcgtcag 5101 agctctgcgc agaatcgcaa atacaccatc aaagtcgagg tgcctaaagg cgcctggcgt 5161 tcgtacttaa atatggaact aaccattcca attttcgcca cgaattccga ctgcgagctt 5221 attgttaagg caatgcaagg tctcctaaaa gatggaaacc cgattccctc agcaatcgca 5281 gcaaactccg gcatctacgg tggcggaggt agcatgtccc atcagaaaat tattcaggat 5341 cttatcgcat ggattgacga gcatattgac cagccgctta acattgatgt agtcgcaaaa 5401 aaatcaggct attcaaagtg gtacttgcaa cgaatgttcc gcacggtgac gcatcagacg 5461 cttggcgatt acattcgcca acgccgcctg ttactggccg ccgttgagtt gcgcaccacc 5521 gagcgtccga tttttgatat cgcaatggac ctgggttatg tctcgcagca gaccttctcc 5581 cgcgttttcg cgcggcagtt tgatcgcact cccgcggatt atcgccaccg cctgtaagcg 5641 gccgccacgc aaaaaacccc gcttcggcgg ggttttttcg ccctagggat atattccgct 5701 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac 5761 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg 5821 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac 5881 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 5941 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt 6001 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc 6061 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac 6121 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt 6181 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac 6241 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca 6301 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac 6361 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat 6421 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt 6481 gttactagat tgacagctag ctcagtccta ggtataatac tagtttgtgt ccagaacgct 6541 ccgtgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 6601 aagtggcaca tgaggatcac ccatgtgctt tttttgaagc ttgggcccga acaaaaactc 6661 atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttaa 6721 acggtctcca gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta 6781 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg 6841 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 6901 ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 6961 aaagactggg cctttcgttt tatctgttgt ttgtcggtga actggatcct tactcgagtc 7021 tagactgcag gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 7081 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 7141 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaaac tagtgcttgg 7201 attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag 7261 ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg 7321 ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga 7381 agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt 7441 gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt 7501 ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa 7561 taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata 7621 tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag 7681 tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt 7741 ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa 7801 aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca 7861 gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt 7921 tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag 7981 cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt 8041 cattatggtg aaagttggaa cctcttacgt gcc //