LOCUS       sequence-305187-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate with sg-2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7459)
  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 7459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2020-06-2"; volume: "11(1):2746. doi"; pages: " 
            10.1038/s41467-020-16574-1"
COMMENT     Sequence Label: pJ2043
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        3..287
                     /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     210..229
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    348..367
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    474..493
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    560..579
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             584..606
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             614..1267
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        1987..2021
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2171..2190
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2195..2217
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2225..2938
                     /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    3358..3377
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3382..3404
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      4631..4658
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4846..5661)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4846..5661)
                     /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     5569..5588
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5662..5753)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5771..6406)
                     /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(5916..5935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6557..7435)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 agaagaaacc aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct
       61 cttctcgcta accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac
      121 caaagccatg acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat
      181 tgattatttg cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc
      241 ggttcctacc tgacgctttt tatcgcaact ctctactgtt tctccatacc gaattcatag
      301 gatagattct ggaaacttta ccgtccgagc tccagcctgc ggtccgggtt cactgccgta
      361 taggcagatc agtgtgtact aagtactgtt tcagagctat gctggaaaca gcatagcaag
      421 ttgaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcgttcact
      481 gccgtatagg cagtcgctgg gacgcccgct cgagcaataa acagttgata gggcttctcc
      541 gttacagcct gcggtccggg ttcactgccg tataggcagt aattttgttt aactttaaga
      601 aggagatata catatggttt cggttatcaa accagagatg aaaatgcgtt actatatgga
      661 tggttcagta aatggtcacg aatttactat tgagggcgag ggtacgggac gcccatacga
      721 ggggcaccag gaaatgactt tacgcgtcac aatggctgaa ggcgggccta tgccgtttgc
      781 gttcgatctt gttagtcatg tcttttgtta cggtcaccgt gtatttacta aataccccga
      841 ggaaattcca gactatttca aacaagcctt cccggaaggt ttgtcttggg agcgcagttt
      901 agagtttgaa gacggtggct cggccagcgt gtcagctcat attagtcttc gcggcaatac
      961 attttatcac aagtcaaagt tcaccggcgt gaacttcccc gcagacggcc caatcatgca
     1021 gaatcaaagt gttgattggg aaccgtccac agagaagatt acagcttccg atggagtctt
     1081 aaagggcgat gtaaccatgt acttaaaatt agaaggggga gggaaccata aatgtcagat
     1141 gaagactacc tataaggccg caaaagagat tcttgaaatg cccggagacc actacattgg
     1201 gcatcgtttg gtccgtaaga cagaaggaaa tattactgaa caggtcgaag acgctgtggc
     1261 acacagcatg tcccgccgta atactgacgc catcacaatc cacagcatcc tggattggat
     1321 tgaagacaac ttggagtcgc cgttgagttt agaaaaagtt agtgaacgta gtggttactc
     1381 aaagtggcac cttcagcgca tgtttaagaa ggaaacgggt cattcattgg gtcaatatat
     1441 tcgttctcgc aagatgactg aaattgccca gaaattgaaa gagtctaatg aacctatttt
     1501 gtacctggcg gagcgttacg gctttgaaag tcagcaaacc cttacacgta ccttcaagaa
     1561 ttactttgac gttccaccac acaaatatcg tatgaccaac atgcagggtg agtcacgttt
     1621 tttgcatccg ttgaatcatt acaattccta ataatcgctg ggacgcccgc catggttcag
     1681 ccaaaaaact taagaccgcc ggtcttgtcc actaccttgc agtaatgcgg tggacaggat
     1741 cggcggtttt cttttctctt ctcaattctt ctgacctgta acgaataata gatagtaaag
     1801 tagtctccga ttgagttttc tctgccgagt cccacccagt tctgtgattt cagtaagttg
     1861 gtaattgata cactgttgcg agaactgctg cctggtagta gataggttgt tattgagtaa
     1921 gaaggtaaag tgaacgaaat ccctgaaact gagactgtag aaaataagct tcagcctgcg
     1981 gtccggttga cagctagctc agtcctaggt actgtgctag ctcgctggga cgcccgggga
     2041 ctacacttac gaaactattg attgctcagc ctgcggtccg gccaagtact tagtacacac
     2101 tgattcgctg ggacgcccgg gatccaagag atttctacac gattgagcac tgtctcagcc
     2161 tgcggtccgg gttcactgcc gtataggcag taattttgtt taactttaag aaggagatat
     2221 acatatgcgt aaaggcgaag aactgtttac cggtgtggtt ccgattctgg tggaactgga
     2281 cggcgatgtt aatggtcata aattcagtgt tcgcggcgaa ggtgaaggcg atgcgacgaa
     2341 cggcaaactg accctgaaat ttatctgcac cacgggtaaa ctgccggtcc cgtggccgac
     2401 gctggtgacc acgctgacct atggcgttca atgttttgcg cgttacccgg atcacatgaa
     2461 acagcacgac tttttcaaat cggccatgcc ggaaggctat gtgcaggaac gtacgattag
     2521 ctttaaagac gatggtacgt ataaaacccg cgcggaagtg aaattcgaag gcgataccct
     2581 ggttaaccgt atcgaactga aaggtatcga tttcaaagaa gacggcaata ttctgggtca
     2641 taaactggaa tataacttca attcccacaa cgtgtacatc accgcggata aacagaaaaa
     2701 cggcattaaa gccaatttca aaatccgcca taatgtggaa gatggtagcg ttcagctggc
     2761 cgaccactat cagcaaaaca cgccgattgg tgatggcccg gtcctgctgc cggacaatca
     2821 ctacctgagt acccagtccg tgctgtcaaa agatccgaac gaaaaacgtg accacatggt
     2881 cctgctggaa tttgtgacgg ctgcgggtat cacccacggc atggacgaac tgtataaaat
     2941 gtcccgccgt aatactgacg ccatcacaat ccacagcatc ctggattgga ttgaagacta
     3001 ataatcgctg ggacgcccgc ctgcaggctc ggtaccaaat tccagaaaag aggcctcccg
     3061 aaaggggggc cttttttcgt tttggtccta atagataaag gataggtctg gtagtgttgt
     3121 tcgttctcgc aggtaaatca ataatactca gcagttccgt agacttttca gtgggacagg
     3181 gtagcgataa cagatagatt gtaataagac acagtaggtg ctcgtagttg cgtgaagaga
     3241 accgctcagg aaatccagtc agaagtattg gtaatcgttg aaaactcagt cgacgcactt
     3301 actgaagacg tcctattaca ctcgtcgttg gaaactgaag atcagcctgc ggtccgggtt
     3361 cactgccgta taggcagtaa ttttgtttaa ctttaagaag gagatataca tatgaatcag
     3421 tcattcatct cggacatctt atatgccgac atcgaatcga aggctaagga acttacagtc
     3481 aattccaaca atactgtcca gccggtcgcg cttatgcgct taggagtttt cgttcccaaa
     3541 ccttccaaga gcaaaggaga aagtaaggaa attgacgcca ccaaagcctt ctctcaactg
     3601 gagattgcta aagcagaggg catggttagt aaaggagaag aaaataacat ggcactgatt
     3661 aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     3721 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggcc
     3781 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     3841 agcaaaacct tcatcaacca cacccagggc atccccgact tctttaagca gtccttccct
     3901 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     3961 caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac
     4021 ttcccatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ctccaccgag
     4081 accctgtacc ccgctgacgg cggcctggaa ggcagagccg acatggccct gaagctcgtg
     4141 ggcgggggcc acctgatctg caacttgaag accacataca gatccaagaa acccgctaag
     4201 aacctcaaga tgcccggcgt ctactatgtg gacagaagac tggaaagaat caaggaggcc
     4261 gacaaagaga cctacgtcga gcagcacgag gtggctgtgg ccagatactg cgacctccct
     4321 agcaaactgg ggcacagagt ctaataatcg ctgggacgcc cggcggccgc ggaaacacag
     4381 aaaaaagccc gcacctgaca gtgcgggctt tttttttcga ccaaaggtag cgaacgacga
     4441 gtcactgttg aggataaata ctttctctac taggcgcctg ttacacaggt cctcagcggc
     4501 gcgcctttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     4561 gttgtgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     4621 caaactaagc agaaggccat cctgacggat ggcctttttg cgtttcagat ctaccggtaa
     4681 accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg acaagctgac
     4741 gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4801 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     4861 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     4921 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     4981 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     5041 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     5101 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     5161 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     5221 aaatacgcgg tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     5281 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     5341 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     5401 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     5461 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     5521 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     5581 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     5641 ttcccgttga atatggctca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5701 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggcat
     5761 gctagcgcag aaacgtccta gaagatgcca ggaggatact tagcagagag acaataaggc
     5821 cggagcgaag ccgtttttcc ataggctccg cccccctgac gaacatcacg aaatctgacg
     5881 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctga
     5941 tggctccctc ttgcgctctc ctgttcccgt cctgcggcgt ccgtgttgtg gtggaggctt
     6001 tacccaaatc accacgtccc gttccgtgta gacagttcgc tccaagctgg gctgtgtgca
     6061 agaacccccc gttcagcccg actgctgcgc cttatccggt aactatcatc ttgagtccaa
     6121 cccggaaaga cacgacaaaa cgccactggc agcagccatt ggtaactgag aattagtgga
     6181 tttagatatc gagagtcttg aagtggtggc ctaacagagg ctacactgaa aggacagtat
     6241 ttggtatctg cgctccacta aagccagtta ccaggttaag cagttcccca actgacttaa
     6301 ccttcgatca aaccgcctcc ccaggcggtt ttttcgttta cagagcagga gattacgacg
     6361 atcgtaaaag gatctcaaga agatccttta cggattcccg acaccatcac tctagatttc
     6421 agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta
     6481 attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gctcctaggg
     6541 tctgattcgt taccaattat gacaacttga cggctacatc attcactttt tcttcacaac
     6601 cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga
     6661 gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca
     6721 aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta
     6781 actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc
     6841 tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa gcctcgcgta
     6901 cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca
     6961 attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt
     7021 taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga
     7081 accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggac
     7141 gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa
     7201 tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct cgtccctgat
     7261 ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc attcccagcg
     7321 gtcggtcgat aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca
     7381 gatgggcatt aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt
     7441 tcatactccc gccattcag
//
