LOCUS       sequence-305188-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate with sg-4.
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: pJ2040
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        84..368
                     /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     291..310
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    429..448
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    555..574
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    641..660
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             665..687
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             695..1348
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2068..2102
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2252..2271
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2276..2298
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2306..3019
                     /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    3439..3458
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3463..3485
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      4712..4739
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4927..5742)
                     /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(4927..5742)
                     /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     5650..5669
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5743..5834)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5852..6487)
                     /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(5997..6016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6638..7459,1..57))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact
       61 tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg
      121 tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag
      181 cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa
      241 tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc
      301 atttttatcc ataagattag cggttcctac ctgacgcttt ttatcgcaac tctctactgt
      361 ttctccatac cgaattcata ggatagattc tggaaacttt accgtccgag ctccagcctg
      421 cggtccgggt tcactgccgt ataggcagga cacatcttag agtatgtagt ttcagagcta
      481 tgctggaaac agcatagcaa gttgaaataa ggctagtccg ttatcaactt gaaaaagtgg
      541 caccgagtcg gtgcgttcac tgccgtatag gcagtcgctg ggacgcccgc tcgagcaata
      601 aacagttgat agggcttctc cgttacagcc tgcggtccgg gttcactgcc gtataggcag
      661 taattttgtt taactttaag aaggagatat acatatggtt tcggttatca aaccagagat
      721 gaaaatgcgt tactatatgg atggttcagt aaatggtcac gaatttacta ttgagggcga
      781 gggtacggga cgcccatacg aggggcacca ggaaatgact ttacgcgtca caatggctga
      841 aggcgggcct atgccgtttg cgttcgatct tgttagtcat gtcttttgtt acggtcaccg
      901 tgtatttact aaataccccg aggaaattcc agactatttc aaacaagcct tcccggaagg
      961 tttgtcttgg gagcgcagtt tagagtttga agacggtggc tcggccagcg tgtcagctca
     1021 tattagtctt cgcggcaata cattttatca caagtcaaag ttcaccggcg tgaacttccc
     1081 cgcagacggc ccaatcatgc agaatcaaag tgttgattgg gaaccgtcca cagagaagat
     1141 tacagcttcc gatggagtct taaagggcga tgtaaccatg tacttaaaat tagaaggggg
     1201 agggaaccat aaatgtcaga tgaagactac ctataaggcc gcaaaagaga ttcttgaaat
     1261 gcccggagac cactacattg ggcatcgttt ggtccgtaag acagaaggaa atattactga
     1321 acaggtcgaa gacgctgtgg cacacagcat gtcccgccgt aatactgacg ccatcacaat
     1381 ccacagcatc ctggattgga ttgaagacaa cttggagtcg ccgttgagtt tagaaaaagt
     1441 tagtgaacgt agtggttact caaagtggca ccttcagcgc atgtttaaga aggaaacggg
     1501 tcattcattg ggtcaatata ttcgttctcg caagatgact gaaattgccc agaaattgaa
     1561 agagtctaat gaacctattt tgtacctggc ggagcgttac ggctttgaaa gtcagcaaac
     1621 ccttacacgt accttcaaga attactttga cgttccacca cacaaatatc gtatgaccaa
     1681 catgcagggt gagtcacgtt ttttgcatcc gttgaatcat tacaattcct aataatcgct
     1741 gggacgcccg ccatggttca gccaaaaaac ttaagaccgc cggtcttgtc cactaccttg
     1801 cagtaatgcg gtggacagga tcggcggttt tcttttctct tctcaattct tctgacctgt
     1861 aacgaataat agatagtaaa gtagtctccg attgagtttt ctctgccgag tcccacccag
     1921 ttctgtgatt tcagtaagtt ggtaattgat acactgttgc gagaactgct gcctggtagt
     1981 agataggttg ttattgagta agaaggtaaa gtgaacgaaa tccctgaaac tgagactgta
     2041 gaaaataagc ttcagcctgc ggtccggttg acagctagct cagtcctagg tactgtgcta
     2101 gctcgctggg acgcccgggg actacactta cgaaactatt gattgctcag cctgcggtcc
     2161 ggccatacat actctaagat gtgtctcgct gggacgcccg ggatccaaga gatttctaca
     2221 cgattgagca ctgtctcagc ctgcggtccg ggttcactgc cgtataggca gtaattttgt
     2281 ttaactttaa gaaggagata tacatatgcg taaaggcgaa gaactgttta ccggtgtggt
     2341 tccgattctg gtggaactgg acggcgatgt taatggtcat aaattcagtg ttcgcggcga
     2401 aggtgaaggc gatgcgacga acggcaaact gaccctgaaa tttatctgca ccacgggtaa
     2461 actgccggtc ccgtggccga cgctggtgac cacgctgacc tatggcgttc aatgttttgc
     2521 gcgttacccg gatcacatga aacagcacga ctttttcaaa tcggccatgc cggaaggcta
     2581 tgtgcaggaa cgtacgatta gctttaaaga cgatggtacg tataaaaccc gcgcggaagt
     2641 gaaattcgaa ggcgataccc tggttaaccg tatcgaactg aaaggtatcg atttcaaaga
     2701 agacggcaat attctgggtc ataaactgga atataacttc aattcccaca acgtgtacat
     2761 caccgcggat aaacagaaaa acggcattaa agccaatttc aaaatccgcc ataatgtgga
     2821 agatggtagc gttcagctgg ccgaccacta tcagcaaaac acgccgattg gtgatggccc
     2881 ggtcctgctg ccggacaatc actacctgag tacccagtcc gtgctgtcaa aagatccgaa
     2941 cgaaaaacgt gaccacatgg tcctgctgga atttgtgacg gctgcgggta tcacccacgg
     3001 catggacgaa ctgtataaaa tgtcccgccg taatactgac gccatcacaa tccacagcat
     3061 cctggattgg attgaagact aataatcgct gggacgcccg cctgcaggct cggtaccaaa
     3121 ttccagaaaa gaggcctccc gaaagggggg ccttttttcg ttttggtcct aatagataaa
     3181 ggataggtct ggtagtgttg ttcgttctcg caggtaaatc aataatactc agcagttccg
     3241 tagacttttc agtgggacag ggtagcgata acagatagat tgtaataaga cacagtaggt
     3301 gctcgtagtt gcgtgaagag aaccgctcag gaaatccagt cagaagtatt ggtaatcgtt
     3361 gaaaactcag tcgacgcact tactgaagac gtcctattac actcgtcgtt ggaaactgaa
     3421 gatcagcctg cggtccgggt tcactgccgt ataggcagta attttgttta actttaagaa
     3481 ggagatatac atatgaatca gtcattcatc tcggacatct tatatgccga catcgaatcg
     3541 aaggctaagg aacttacagt caattccaac aatactgtcc agccggtcgc gcttatgcgc
     3601 ttaggagttt tcgttcccaa accttccaag agcaaaggag aaagtaagga aattgacgcc
     3661 accaaagcct tctctcaact ggagattgct aaagcagagg gcatggttag taaaggagaa
     3721 gaaaataaca tggcactgat taaggagaac atgcacatga agctgtacat ggagggcacc
     3781 gtgaacaacc accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc
     3841 cagaccatga gaatcaaggc cgtcgagggc ggccctctcc ccttcgcctt cgacatcctg
     3901 gctaccagct tcatgtacgg cagcaaaacc ttcatcaacc acacccaggg catccccgac
     3961 ttctttaagc agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac
     4021 gggggcgtgc tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac
     4081 gtcaagatca gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc
     4141 ggctgggagg cctccaccga gaccctgtac cccgctgacg gcggcctgga aggcagagcc
     4201 gacatggccc tgaagctcgt gggcgggggc cacctgatct gcaacttgaa gaccacatac
     4261 agatccaaga aacccgctaa gaacctcaag atgcccggcg tctactatgt ggacagaaga
     4321 ctggaaagaa tcaaggaggc cgacaaagag acctacgtcg agcagcacga ggtggctgtg
     4381 gccagatact gcgacctccc tagcaaactg gggcacagag tctaataatc gctgggacgc
     4441 ccggcggccg cggaaacaca gaaaaaagcc cgcacctgac agtgcgggct ttttttttcg
     4501 accaaaggta gcgaacgacg agtcactgtt gaggataaat actttctcta ctaggcgcct
     4561 gttacacagg tcctcagcgg cgcgcctttg tcggtgaacg ctctcctgag taggacaaat
     4621 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     4681 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt
     4741 gcgtttcaga tctaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc
     4801 ctgaaccgac gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg
     4861 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     4921 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     4981 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     5041 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     5101 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     5161 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     5221 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     5281 tgattgcgcc tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg
     5341 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     5401 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     5461 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     5521 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     5581 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     5641 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     5701 tcgcggccta gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata
     5761 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5821 gaaaaataaa caaataggca tgctagcgca gaaacgtcct agaagatgcc aggaggatac
     5881 ttagcagaga gacaataagg ccggagcgaa gccgtttttc cataggctcc gcccccctga
     5941 cgaacatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     6001 ataccaggcg tttccccctg atggctccct cttgcgctct cctgttcccg tcctgcggcg
     6061 tccgtgttgt ggtggaggct ttacccaaat caccacgtcc cgttccgtgt agacagttcg
     6121 ctccaagctg ggctgtgtgc aagaaccccc cgttcagccc gactgctgcg ccttatccgg
     6181 taactatcat cttgagtcca acccggaaag acacgacaaa acgccactgg cagcagccat
     6241 tggtaactga gaattagtgg atttagatat cgagagtctt gaagtggtgg cctaacagag
     6301 gctacactga aaggacagta tttggtatct gcgctccact aaagccagtt accaggttaa
     6361 gcagttcccc aactgactta accttcgatc aaaccgcctc cccaggcggt tttttcgttt
     6421 acagagcagg agattacgac gatcgtaaaa ggatctcaag aagatccttt acggattccc
     6481 gacaccatca ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag
     6541 ccccatacga tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag
     6601 ctgactgggt tgctcctagg gtctgattcg ttaccaatta tgacaacttg acggctacat
     6661 cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt
     6721 taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga
     6781 taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc
     6841 agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca
     6901 agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga
     6961 tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg
     7021 cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc
     7081 gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt
     7141 gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt
     7201 catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg
     7261 gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc
     7321 aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa
     7381 tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa
     7441 tcggcgttaa acccgccac
//
