LOCUS Exported 7295 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Control ("C") lacking sg-1. ACCESSION . VERSION . KEYWORDS pJ2044_N2only SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7295) AUTHORS Santos-Moreno J, Tasiudi E, Stelling J, Schaerli Y TITLE Multistable and dynamic CRISPRi-based synthetic circuits. JOURNAL Nat Commun. 2020 Jun 2;11(1):2746. doi: 10.1038/s41467-020-16574-1. PUBMED 32488086 REFERENCE 2 (bases 1 to 7295) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7295 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 27..311 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 234..253 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" protein_bind 425..439 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" RBS 444..466 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 474..1127 /codon_start=1 /product="monomeric Kusabira-Orange 2 fluorescent protein" /label=mKO2 /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS" protein_bind 2036..2050 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" RBS 2055..2077 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2085..2798 /codon_start=1 /product="GFP variant that folds robustly even when fused to poorly folded proteins (Pedelacq et al., 2006)" /label=superfolder GFP /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE FVTAAGITHGMDELYK" protein_bind 3223..3237 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" RBS 3242..3264 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" terminator 4491..4518 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" CDS complement(4706..5521) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 5429..5448 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(5522..5613) /gene="bla" /label=AmpR promoter rep_origin complement(5631..6266) /direction=LEFT /label=ColA ori /note="Plasmids containing the ColA origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins." primer_bind complement(5776..5795) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6417..7295) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" ORIGIN 1 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg 61 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa 121 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta 181 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat 241 agcattttta tccataagat tagcggttcc tacctgacgc tttttatcgc aactctctac 301 tgtttctcca taccgaattc ataggataga ttctggaaac tttaccgtcc gagctcaggc 361 ttaccttact cgagcaataa acagttgata gggcttctcc gttacagcct gcggtccggg 421 ttcactgccg tataggcagt aattttgttt aactttaaga aggagatata catatggttt 481 cggttatcaa accagagatg aaaatgcgtt actatatgga tggttcagta aatggtcacg 541 aatttactat tgagggcgag ggtacgggac gcccatacga ggggcaccag gaaatgactt 601 tacgcgtcac aatggctgaa ggcgggccta tgccgtttgc gttcgatctt gttagtcatg 661 tcttttgtta cggtcaccgt gtatttacta aataccccga ggaaattcca gactatttca 721 aacaagcctt cccggaaggt ttgtcttggg agcgcagttt agagtttgaa gacggtggct 781 cggccagcgt gtcagctcat attagtcttc gcggcaatac attttatcac aagtcaaagt 841 tcaccggcgt gaacttcccc gcagacggcc caatcatgca gaatcaaagt gttgattggg 901 aaccgtccac agagaagatt acagcttccg atggagtctt aaagggcgat gtaaccatgt 961 acttaaaatt agaaggggga gggaaccata aatgtcagat gaagactacc tataaggccg 1021 caaaagagat tcttgaaatg cccggagacc actacattgg gcatcgtttg gtccgtaaga 1081 cagaaggaaa tattactgaa caggtcgaag acgctgtggc acacagcatg tcccgccgta 1141 atactgacgc catcacaatc cacagcatcc tggattggat tgaagacaac ttggagtcgc 1201 cgttgagttt agaaaaagtt agtgaacgta gtggttactc aaagtggcac cttcagcgca 1261 tgtttaagaa ggaaacgggt cattcattgg gtcaatatat tcgttctcgc aagatgactg 1321 aaattgccca gaaattgaaa gagtctaatg aacctatttt gtacctggcg gagcgttacg 1381 gctttgaaag tcagcaaacc cttacacgta ccttcaagaa ttactttgac gttccaccac 1441 acaaatatcg tatgaccaac atgcagggtg agtcacgttt tttgcatccg ttgaatcatt 1501 acaattccta ataatcgctg ggacgcccgc catggttcag ccaaaaaact taagaccgcc 1561 ggtcttgtcc actaccttgc agtaatgcgg tggacaggat cggcggtttt cttttctctt 1621 ctcaattctt ctgacctgta acgaataata gatagtaaag tagtctccga ttgagttttc 1681 tctgccgagt cccacccagt tctgtgattt cagtaagttg gtaattgata cactgttgcg 1741 agaactgctg cctggtagta gataggttgt tattgagtaa gaaggtaaag tgaacgaaat 1801 ccctgaaact gagactgtag aaaataagct tcagcctgcg gtccggttga cagctagctc 1861 agtcctaggt actgtgctag ctcgctggga cgcccgggga ctacacttac gaaactattg 1921 attgctcagc ctgcggtccg gccacactag agtctagctt gagatcgctg ggacgcccgg 1981 gatccaagag atttctacac gattgagcac tgtctcagcc tgcggtccgg gttcactgcc 2041 gtataggcag taattttgtt taactttaag aaggagatat acatatgcgt aaaggcgaag 2101 aactgtttac cggtgtggtt ccgattctgg tggaactgga cggcgatgtt aatggtcata 2161 aattcagtgt tcgcggcgaa ggtgaaggcg atgcgacgaa cggcaaactg accctgaaat 2221 ttatctgcac cacgggtaaa ctgccggtcc cgtggccgac gctggtgacc acgctgacct 2281 atggcgttca atgttttgcg cgttacccgg atcacatgaa acagcacgac tttttcaaat 2341 cggccatgcc ggaaggctat gtgcaggaac gtacgattag ctttaaagac gatggtacgt 2401 ataaaacccg cgcggaagtg aaattcgaag gcgataccct ggttaaccgt atcgaactga 2461 aaggtatcga tttcaaagaa gacggcaata ttctgggtca taaactggaa tataacttca 2521 attcccacaa cgtgtacatc accgcggata aacagaaaaa cggcattaaa gccaatttca 2581 aaatccgcca taatgtggaa gatggtagcg ttcagctggc cgaccactat cagcaaaaca 2641 cgccgattgg tgatggcccg gtcctgctgc cggacaatca ctacctgagt acccagtccg 2701 tgctgtcaaa agatccgaac gaaaaacgtg accacatggt cctgctggaa tttgtgacgg 2761 ctgcgggtat cacccacggc atggacgaac tgtataaaat gtcccgccgt aatactgacg 2821 ccatcacaat ccacagcatc ctggattgga ttgaagacta ataatcgctg ggacgcccgc 2881 ctgcaggctc ggtaccaaat tccagaaaag aggcctcccg aaaggggggc cttttttcgt 2941 tttggtccta atagataaag gataggtctg gtagtgttgt tcgttctcgc aggtaaatca 3001 ataatactca gcagttccgt agacttttca gtgggacagg gtagcgataa cagatagatt 3061 gtaataagac acagtaggtg ctcgtagttg cgtgaagaga accgctcagg aaatccagtc 3121 agaagtattg gtaatcgttg aaaactcagt cgacgcactt actgaagacg tcctattaca 3181 ctcgtcgttg gaaactgaag atcagcctgc ggtccgggtt cactgccgta taggcagtaa 3241 ttttgtttaa ctttaagaag gagatataca tatgaatcag tcattcatct cggacatctt 3301 atatgccgac atcgaatcga aggctaagga acttacagtc aattccaaca atactgtcca 3361 gccggtcgcg cttatgcgct taggagtttt cgttcccaaa ccttccaaga gcaaaggaga 3421 aagtaaggaa attgacgcca ccaaagcctt ctctcaactg gagattgcta aagcagaggg 3481 catggttagt aaaggagaag aaaataacat ggcactgatt aaggagaaca tgcacatgaa 3541 gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg 3601 caagccctac gagggcaccc agaccatgag aatcaaggcc gtcgagggcg gccctctccc 3661 cttcgccttc gacatcctgg ctaccagctt catgtacggc agcaaaacct tcatcaacca 3721 cacccagggc atccccgact tctttaagca gtccttccct gagggcttca catgggagag 3781 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga 3841 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt 3901 gatgcagaag aaaacactcg gctgggaggc ctccaccgag accctgtacc ccgctgacgg 3961 cggcctggaa ggcagagccg acatggccct gaagctcgtg ggcgggggcc acctgatctg 4021 caacttgaag accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt 4081 ctactatgtg gacagaagac tggaaagaat caaggaggcc gacaaagaga cctacgtcga 4141 gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacagagt 4201 ctaataatcg ctgggacgcc cggcggccgc ggaaacacag aaaaaagccc gcacctgaca 4261 gtgcgggctt tttttttcga ccaaaggtag cgaacgacga gtcactgttg aggataaata 4321 ctttctctac taggcgcctg ttacacaggt cctcagcggc gcgcctttgt cggtgaacgc 4381 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg 4441 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat 4501 cctgacggat ggcctttttg cgtttcagat ctaccggtaa accagcaata gacataagcg 4561 gctatttaac gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg 4621 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 4681 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca 4741 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 4801 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 4861 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 4921 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt 4981 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 5041 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa 5101 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 5161 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 5221 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 5281 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 5341 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 5401 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 5461 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca 5521 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 5581 acatatttga atgtatttag aaaaataaac aaataggcat gctagcgcag aaacgtccta 5641 gaagatgcca ggaggatact tagcagagag acaataaggc cggagcgaag ccgtttttcc 5701 ataggctccg cccccctgac gaacatcacg aaatctgacg ctcaaatcag tggtggcgaa 5761 acccgacagg actataaaga taccaggcgt ttccccctga tggctccctc ttgcgctctc 5821 ctgttcccgt cctgcggcgt ccgtgttgtg gtggaggctt tacccaaatc accacgtccc 5881 gttccgtgta gacagttcgc tccaagctgg gctgtgtgca agaacccccc gttcagcccg 5941 actgctgcgc cttatccggt aactatcatc ttgagtccaa cccggaaaga cacgacaaaa 6001 cgccactggc agcagccatt ggtaactgag aattagtgga tttagatatc gagagtcttg 6061 aagtggtggc ctaacagagg ctacactgaa aggacagtat ttggtatctg cgctccacta 6121 aagccagtta ccaggttaag cagttcccca actgacttaa ccttcgatca aaccgcctcc 6181 ccaggcggtt ttttcgttta cagagcagga gattacgacg atcgtaaaag gatctcaaga 6241 agatccttta cggattcccg acaccatcac tctagatttc agtgcaattt atctcttcaa 6301 atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc 6361 ccgcgcccac cggaaggagc tgactgggtt gctcctaggg tctgattcgt taccaattat 6421 gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg 6481 ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca 6541 ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg 6601 atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt 6661 gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg 6721 tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga 6781 tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc 6841 gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg 6901 tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt 6961 gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga 7021 taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc 7081 aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg 7141 cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg 7201 agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt aaacgagtat 7261 cccggcagca ggggatcatt ttgcgcttca gccat //