LOCUS       sequence-305186-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate with sg-1.
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: pJ2044
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    84..103
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    210..229
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    296..315
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             320..342
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             350..1003
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        1723..1757
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    1907..1926
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1931..1953
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1961..2674
                     /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    3094..3113
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3118..3140
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      4367..4394
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4582..5397)
                     /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(4582..5397)
                     /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     5305..5324
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5398..5489)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5507..6142)
                     /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(5652..5671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6293..7171)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(7198..7459,1..23)
                     /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     7405..7424
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 gcaactctct actgtttctc cataccgaat tcataggata gattctggaa actttaccgt
       61 ccgagctcca gcctgcggtc cgggttcact gccgtatagg cagtctcaag ctagactcta
      121 gtggtttcag agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc
      181 aacttgaaaa agtggcaccg agtcggtgcg ttcactgccg tataggcagt cgctgggacg
      241 cccgctcgag caataaacag ttgatagggc ttctccgtta cagcctgcgg tccgggttca
      301 ctgccgtata ggcagtaatt ttgtttaact ttaagaagga gatatacata tggtttcggt
      361 tatcaaacca gagatgaaaa tgcgttacta tatggatggt tcagtaaatg gtcacgaatt
      421 tactattgag ggcgagggta cgggacgccc atacgagggg caccaggaaa tgactttacg
      481 cgtcacaatg gctgaaggcg ggcctatgcc gtttgcgttc gatcttgtta gtcatgtctt
      541 ttgttacggt caccgtgtat ttactaaata ccccgaggaa attccagact atttcaaaca
      601 agccttcccg gaaggtttgt cttgggagcg cagtttagag tttgaagacg gtggctcggc
      661 cagcgtgtca gctcatatta gtcttcgcgg caatacattt tatcacaagt caaagttcac
      721 cggcgtgaac ttccccgcag acggcccaat catgcagaat caaagtgttg attgggaacc
      781 gtccacagag aagattacag cttccgatgg agtcttaaag ggcgatgtaa ccatgtactt
      841 aaaattagaa gggggaggga accataaatg tcagatgaag actacctata aggccgcaaa
      901 agagattctt gaaatgcccg gagaccacta cattgggcat cgtttggtcc gtaagacaga
      961 aggaaatatt actgaacagg tcgaagacgc tgtggcacac agcatgtccc gccgtaatac
     1021 tgacgccatc acaatccaca gcatcctgga ttggattgaa gacaacttgg agtcgccgtt
     1081 gagtttagaa aaagttagtg aacgtagtgg ttactcaaag tggcaccttc agcgcatgtt
     1141 taagaaggaa acgggtcatt cattgggtca atatattcgt tctcgcaaga tgactgaaat
     1201 tgcccagaaa ttgaaagagt ctaatgaacc tattttgtac ctggcggagc gttacggctt
     1261 tgaaagtcag caaaccctta cacgtacctt caagaattac tttgacgttc caccacacaa
     1321 atatcgtatg accaacatgc agggtgagtc acgttttttg catccgttga atcattacaa
     1381 ttcctaataa tcgctgggac gcccgccatg gttcagccaa aaaacttaag accgccggtc
     1441 ttgtccacta ccttgcagta atgcggtgga caggatcggc ggttttcttt tctcttctca
     1501 attcttctga cctgtaacga ataatagata gtaaagtagt ctccgattga gttttctctg
     1561 ccgagtccca cccagttctg tgatttcagt aagttggtaa ttgatacact gttgcgagaa
     1621 ctgctgcctg gtagtagata ggttgttatt gagtaagaag gtaaagtgaa cgaaatccct
     1681 gaaactgaga ctgtagaaaa taagcttcag cctgcggtcc ggttgacagc tagctcagtc
     1741 ctaggtactg tgctagctcg ctgggacgcc cggggactac acttacgaaa ctattgattg
     1801 ctcagcctgc ggtccggcca cactagagtc tagcttgaga tcgctgggac gcccgggatc
     1861 caagagattt ctacacgatt gagcactgtc tcagcctgcg gtccgggttc actgccgtat
     1921 aggcagtaat tttgtttaac tttaagaagg agatatacat atgcgtaaag gcgaagaact
     1981 gtttaccggt gtggttccga ttctggtgga actggacggc gatgttaatg gtcataaatt
     2041 cagtgttcgc ggcgaaggtg aaggcgatgc gacgaacggc aaactgaccc tgaaatttat
     2101 ctgcaccacg ggtaaactgc cggtcccgtg gccgacgctg gtgaccacgc tgacctatgg
     2161 cgttcaatgt tttgcgcgtt acccggatca catgaaacag cacgactttt tcaaatcggc
     2221 catgccggaa ggctatgtgc aggaacgtac gattagcttt aaagacgatg gtacgtataa
     2281 aacccgcgcg gaagtgaaat tcgaaggcga taccctggtt aaccgtatcg aactgaaagg
     2341 tatcgatttc aaagaagacg gcaatattct gggtcataaa ctggaatata acttcaattc
     2401 ccacaacgtg tacatcaccg cggataaaca gaaaaacggc attaaagcca atttcaaaat
     2461 ccgccataat gtggaagatg gtagcgttca gctggccgac cactatcagc aaaacacgcc
     2521 gattggtgat ggcccggtcc tgctgccgga caatcactac ctgagtaccc agtccgtgct
     2581 gtcaaaagat ccgaacgaaa aacgtgacca catggtcctg ctggaatttg tgacggctgc
     2641 gggtatcacc cacggcatgg acgaactgta taaaatgtcc cgccgtaata ctgacgccat
     2701 cacaatccac agcatcctgg attggattga agactaataa tcgctgggac gcccgcctgc
     2761 aggctcggta ccaaattcca gaaaagaggc ctcccgaaag gggggccttt tttcgttttg
     2821 gtcctaatag ataaaggata ggtctggtag tgttgttcgt tctcgcaggt aaatcaataa
     2881 tactcagcag ttccgtagac ttttcagtgg gacagggtag cgataacaga tagattgtaa
     2941 taagacacag taggtgctcg tagttgcgtg aagagaaccg ctcaggaaat ccagtcagaa
     3001 gtattggtaa tcgttgaaaa ctcagtcgac gcacttactg aagacgtcct attacactcg
     3061 tcgttggaaa ctgaagatca gcctgcggtc cgggttcact gccgtatagg cagtaatttt
     3121 gtttaacttt aagaaggaga tatacatatg aatcagtcat tcatctcgga catcttatat
     3181 gccgacatcg aatcgaaggc taaggaactt acagtcaatt ccaacaatac tgtccagccg
     3241 gtcgcgctta tgcgcttagg agttttcgtt cccaaacctt ccaagagcaa aggagaaagt
     3301 aaggaaattg acgccaccaa agccttctct caactggaga ttgctaaagc agagggcatg
     3361 gttagtaaag gagaagaaaa taacatggca ctgattaagg agaacatgca catgaagctg
     3421 tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag
     3481 ccctacgagg gcacccagac catgagaatc aaggccgtcg agggcggccc tctccccttc
     3541 gccttcgaca tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc
     3601 cagggcatcc ccgacttctt taagcagtcc ttccctgagg gcttcacatg ggagagagtc
     3661 accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc
     3721 tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg
     3781 cagaagaaaa cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc
     3841 ctggaaggca gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac
     3901 ttgaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac
     3961 tatgtggaca gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag
     4021 cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca cagagtctaa
     4081 taatcgctgg gacgcccggc ggccgcggaa acacagaaaa aagcccgcac ctgacagtgc
     4141 gggctttttt tttcgaccaa aggtagcgaa cgacgagtca ctgttgagga taaatacttt
     4201 ctctactagg cgcctgttac acaggtcctc agcggcgcgc ctttgtcggt gaacgctctc
     4261 ctgagtagga caaatccgcc gggagcggat ttgaacgttg tgaagcaacg gcccggaggg
     4321 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ctaagcagaa ggccatcctg
     4381 acggatggcc tttttgcgtt tcagatctac cggtaaacca gcaatagaca taagcggcta
     4441 tttaacgacc ctgccctgaa ccgacgacaa gctgacgacc gggtctccgc aagtggcact
     4501 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4561 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     4621 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     4681 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     4741 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     4801 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     4861 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     4921 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg
     4981 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     5041 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     5101 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     5161 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     5221 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     5281 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     5341 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcatact
     5401 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5461 atttgaatgt atttagaaaa ataaacaaat aggcatgcta gcgcagaaac gtcctagaag
     5521 atgccaggag gatacttagc agagagacaa taaggccgga gcgaagccgt ttttccatag
     5581 gctccgcccc cctgacgaac atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
     5641 gacaggacta taaagatacc aggcgtttcc ccctgatggc tccctcttgc gctctcctgt
     5701 tcccgtcctg cggcgtccgt gttgtggtgg aggctttacc caaatcacca cgtcccgttc
     5761 cgtgtagaca gttcgctcca agctgggctg tgtgcaagaa ccccccgttc agcccgactg
     5821 ctgcgcctta tccggtaact atcatcttga gtccaacccg gaaagacacg acaaaacgcc
     5881 actggcagca gccattggta actgagaatt agtggattta gatatcgaga gtcttgaagt
     5941 ggtggcctaa cagaggctac actgaaagga cagtatttgg tatctgcgct ccactaaagc
     6001 cagttaccag gttaagcagt tccccaactg acttaacctt cgatcaaacc gcctccccag
     6061 gcggtttttt cgtttacaga gcaggagatt acgacgatcg taaaaggatc tcaagaagat
     6121 cctttacgga ttcccgacac catcactcta gatttcagtg caatttatct cttcaaatgt
     6181 agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc
     6241 gcccaccgga aggagctgac tgggttgctc ctagggtctg attcgttacc aattatgaca
     6301 acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg
     6361 ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc
     6421 gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac
     6481 gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca
     6541 gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg
     6601 ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga
     6661 gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca
     6721 gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc
     6781 tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg
     6841 gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc
     6901 attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa
     6961 tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa
     7021 tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat
     7081 aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg
     7141 gcagcagggg atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag
     7201 aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct
     7261 cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag
     7321 ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt
     7381 atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggttc
     7441 ctacctgacg ctttttatc
//
