LOCUS Exported 6900 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION CRISPRlator control ("open ring"). ACCESSION . VERSION . KEYWORDS pJ2072.2_c1 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6900) 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 6900) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6900 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 241..255 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" protein_bind 367..381 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" protein_bind 453..467 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" RBS 472..494 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 502..1209 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(647..665) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 915..934 /label=mCherry-F /note="mCherry, forward primer" protein_bind 2112..2126 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" protein_bind 2238..2252 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" protein_bind 2324..2338 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" RBS 2343..2365 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" protein_bind 3641..3655 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" protein_bind 3767..3781 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" protein_bind 3853..3867 /label=Csy4 site /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)" /note="cleavage site for the Csy4 endoribonuclease (Haurwitz et al., 2010)" RBS 3872..3894 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" primer_bind complement(4142..4170) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 4765..4785 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" terminator 5115..5142 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" CDS complement(5330..6145) /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 6053..6072 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(6146..6237) /gene="bla" /label=AmpR promoter rep_origin complement(6255..6890) /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(6400..6419) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gaattcatag gatagattct ggaaacttta ccgtccgagc tccagcctgc ggtccggttg 61 acggctagct cagtcctagg tacagtgcta gctcgctggg acgcccgaga gccgaatcgc 121 acttatttac agtagttcag cctgcggtcc ggccatacat actctaagat gtgtctcgct 181 gggacgcccg gggactacac ttacgaaact attgattgct cagcctgcgg tccgggttca 241 ctgccgtata ggcagatcag tgtgtactaa gtactgtttc agagctatgc tggaaacagc 301 atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 361 cgttcactgc cgtataggca gtcgctggga cgcccgctcg agcaataaac agttgatagg 421 gcttctccgt tacagcctgc ggtccgggtt cactgccgta taggcagtaa ttttgtttaa 481 ctttaagaag gagatataca tatggtgagc aagggcgagg aggataacat ggccatcatc 541 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag 601 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg 661 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc 721 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc 781 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc 841 caggactcct ctctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac 901 ttcccctccg acggccccgt aatgcagaag aagactatgg gctgggaggc ctcctccgag 961 cggatgtacc ccgaggacgg cgcgctgaag ggcgagatca agcagaggct gaagctgaag 1021 gacggcggcc actacgacgc tgaagtcaag accacctaca aggccaagaa gcccgtgcag 1081 ctgcccggcg cgtacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac 1141 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag 1201 ctgtacaaga tgtcccgccg taatactgac gccatcacaa tccacagcat cctggattgg 1261 attgaagaca acttggagtc gccgttgagt ttagaaaaag ttagtgaacg tagtggttac 1321 tcaaagtggc accttcagcg catgtttaag aaggaaacgg gtcattcatt gggtcaatat 1381 attcgttctc gcaagatgac tgaaattgcc cagaaattga aagagtctaa tgaacctatt 1441 ttgtacctgg cggagcgtta cggctttgaa agtcagcaaa cccttacacg taccttcaag 1501 aattactttg acgttccacc acacaaatat cgtatgacca acatgcaggg tgagtcacgt 1561 tttttgcatc cgttgaatca ttacaattcc taataatcgc tgggacgccc gccatggttc 1621 agccaaaaaa cttaagaccg ccggtcttgt ccactacctt gcagtaatgc ggtggacagg 1681 atcggcggtt ttcttttctc ttctcaattc ttctgacctg taacgaataa tagatagtaa 1741 agtagtctcc gattgagttt tctctgccga gtcccaccca gttctgtgat ttcagtaagt 1801 tggtaattga tacactgttg cgagaactgc tgcctggtag tagataggtt gttattgagt 1861 aagaaggtaa agtgaacgaa atccctgaaa ctgagactgt agaaaataag cttcagcctg 1921 cggtccggtt gacggctagc tcagtcctag gtacagtgct agctcgctgg gacgcccgag 1981 atagccgtta cacaggtgac acttatttca gcctgcggtc cggccacact agagtctagc 2041 ttgagatcgc tgggacgccc gtgaacagtt gctctgattg aaaccacgat tcagcctgcg 2101 gtccgggttc actgccgtat aggcagtgac tgagctagtg tactctgttt cagagctatg 2161 ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca 2221 ccgagtcggt gcgttcactg ccgtataggc agtcgctggg acgcccggga tccaagagat 2281 ttctacacga ttgagcactg tctcagcctg cggtccgggt tcactgccgt ataggcagta 2341 attttgttta actttaagaa ggagatatac atatgtctaa aggtgaagaa ttattcactg 2401 gtgttgtccc aattttggtt gaattagatg gtgatgttaa tggtcacaaa ttttctgtct 2461 ccggtgaagg tgaaggtgat gctacttacg gtaaattgac cttaaaattt atttgtacta 2521 ctggtaaatt gccagttccg tggccaacct tagtcactac tttaggttat ggtttgatgt 2581 gttttgctag atacccagat catatgaaac aacatgactt tttcaagtct gccatgccag 2641 aaggttatgt tcaagaaaga actatttttt tcaaagatga cggtaactac aagaccagag 2701 ctgaagtcaa gtttgaaggt gataccttag ttaatagaat cgaattaaaa ggtattgatt 2761 ttaaagaaga tggtaacatt ttaggtcaca aattggaata caactataac tctcacaatg 2821 tttacatcat ggctgacaaa caaaagaatg gtatcaaagt taacttcaaa attagacaca 2881 acattgaaga tggttctgtt caattagctg accattatca acaaaatact ccaattggtg 2941 atggtccagt cttgttacca gacaaccatt acttatccta tcaatctaaa ttatccaaag 3001 atccaaacga aaagagagac cacatggtct tgttagaatt tgttactgct gctggtatta 3061 cccacggtat ggatgaattg tacaaaatgt cccgccgtaa tactgacgcc atcacaatcc 3121 acagcatcct ggattggatt gaagactaat aatcgctggg acgcccgcct gcaggctcgg 3181 taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtcctaat 3241 agataaagga taggtctggt agtgttgttc gttctcgcag gtaaatcaat aatactcagc 3301 agttccgtag acttttcagt gggacagggt agcgataaca gatagattgt aataagacac 3361 agtaggtgct cgtagttgcg tgaagagaac cgctcaggaa atccagtcag aagtattggt 3421 aatcgttgaa aactcagtcg accagcctgc ggtccggttg acggctagct cagtcctagg 3481 tacagtgcta gctcgctggg acgcccgagt gacgactgcg aagtaacctc tatttatcag 3541 cctgcggtcc ggccaagtac ttagtacaca ctgattcgct gggacgcccg gtagtgctta 3601 tcagacccaa tactgttgaa cagcctgcgg tccgggttca ctgccgtata ggcagtctca 3661 agctagactc tagtggtttc agagctatgc tggaaacagc atagcaagtt gaaataaggc 3721 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cgttcactgc cgtataggca 3781 gtcgctggga cgcccggacg tcctattaca ctcgtcgttg gaaactgaag atcagcctgc 3841 ggtccgggtt cactgccgta taggcagtaa ttttgtttaa ctttaagaag gagatataca 3901 tatgaatcag tcattcatct cggacatctt atatgccgac atcgaatcga aggctaagga 3961 acttacagtc aattccaaca atactgtcca gccggtcgcg cttatgcgct taggagtttt 4021 cgttcccaaa ccttccaaga gcaaaggaga aagtaaggaa attgacgcca ccaaagcctt 4081 ctctcaactg gagattgcta aagcagaggg catgagtaaa ggagaagaac ttttcactgg 4141 agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag 4201 tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac 4261 tggaaaacta cctgttccgt ggccaacact tgtcactact ttgacttggg gtgttcaatg 4321 ctttgctaga tacccagatc atatgaaaca gcatgacttt ttcaagagtg ccatgcccga 4381 aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc 4441 tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt 4501 taaagaagat ggaaacattc ttggacacaa attggaatac aacgctattt cagataatgt 4561 atacatcact gcagacaaac aaaagaatgg aatcaaagct aatttcaaaa ttagacacaa 4621 cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc caattggcga 4681 tggccctgtc cttttaccag acaaccatta cctgtccaca caatctaaac tttcgaaaga 4741 tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac 4801 actaggcatg gatgaactat acaaataata atcgctggga cgcccggcgg ccgcggaaac 4861 acagaaaaaa gcccgcacct gacagtgcgg gctttttttt tcgaccaaag gtagcgaacg 4921 acgagtcact gttgaggata aatactttct ctactaggcg cctgttacac aggtcctcag 4981 cggcgcgcct ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt 5041 gaacgttgtg aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag 5101 gcatcaaact aagcagaagg ccatcctgac ggatggcctt tttgcgtttc agatctaccg 5161 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgacaagc 5221 tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 5281 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact 5341 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt 5401 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa 5461 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc 5521 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg 5581 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct 5641 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga 5701 gacgaaatac gcggtcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc 5761 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata 5821 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac 5881 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca 5941 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg 6001 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag 6061 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag 6121 acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc 6181 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 6241 gcatgctagc gcagaaacgt cctagaagat gccaggagga tacttagcag agagacaata 6301 aggccggagc gaagccgttt ttccataggc tccgcccccc tgacgaacat cacgaaatct 6361 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6421 ctgatggctc cctcttgcgc tctcctgttc ccgtcctgcg gcgtccgtgt tgtggtggag 6481 gctttaccca aatcaccacg tcccgttccg tgtagacagt tcgctccaag ctgggctgtg 6541 tgcaagaacc ccccgttcag cccgactgct gcgccttatc cggtaactat catcttgagt 6601 ccaacccgga aagacacgac aaaacgccac tggcagcagc cattggtaac tgagaattag 6661 tggatttaga tatcgagagt cttgaagtgg tggcctaaca gaggctacac tgaaaggaca 6721 gtatttggta tctgcgctcc actaaagcca gttaccaggt taagcagttc cccaactgac 6781 ttaaccttcg atcaaaccgc ctccccaggc ggttttttcg tttacagagc aggagattac 6841 gacgatcgta aaaggatctc aagaagatcc tttacggatt cccgacacca tcactctaga //