LOCUS       sequence-305159-        7459 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate with sg-3.
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: pJ2039
FEATURES             Location/Qualifiers
     source          1..7459
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..310
                     /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     233..252
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    371..390
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    497..516
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    583..602
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             607..629
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             637..1290
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2010..2044
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2194..2213
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2218..2240
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2248..2961
                     /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    3381..3400
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3405..3427
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      4654..4681
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4869..5684)
                     /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(4869..5684)
                     /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     5592..5611
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5685..5776)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5794..6429)
                     /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(5939..5958)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6580..7458)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 cttttcatac tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc
       61 cgtcactgcg tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa
      121 agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat
      181 aatcacggca gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata
      241 gcatttttat ccataagatt agcggttcct acctgacgct ttttatcgca actctctact
      301 gtttctccat accgaattca taggatagat tctggaaact ttaccgtccg agctccagcc
      361 tgcggtccgg gttcactgcc gtataggcag tgactgagct agtgtactct gtttcagagc
      421 tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac ttgaaaaagt
      481 ggcaccgagt cggtgcgttc actgccgtat aggcagtcgc tgggacgccc gctcgagcaa
      541 taaacagttg atagggcttc tccgttacag cctgcggtcc gggttcactg ccgtataggc
      601 agtaattttg tttaacttta agaaggagat atacatatgg tttcggttat caaaccagag
      661 atgaaaatgc gttactatat ggatggttca gtaaatggtc acgaatttac tattgagggc
      721 gagggtacgg gacgcccata cgaggggcac caggaaatga ctttacgcgt cacaatggct
      781 gaaggcgggc ctatgccgtt tgcgttcgat cttgttagtc atgtcttttg ttacggtcac
      841 cgtgtattta ctaaataccc cgaggaaatt ccagactatt tcaaacaagc cttcccggaa
      901 ggtttgtctt gggagcgcag tttagagttt gaagacggtg gctcggccag cgtgtcagct
      961 catattagtc ttcgcggcaa tacattttat cacaagtcaa agttcaccgg cgtgaacttc
     1021 cccgcagacg gcccaatcat gcagaatcaa agtgttgatt gggaaccgtc cacagagaag
     1081 attacagctt ccgatggagt cttaaagggc gatgtaacca tgtacttaaa attagaaggg
     1141 ggagggaacc ataaatgtca gatgaagact acctataagg ccgcaaaaga gattcttgaa
     1201 atgcccggag accactacat tgggcatcgt ttggtccgta agacagaagg aaatattact
     1261 gaacaggtcg aagacgctgt ggcacacagc atgtcccgcc gtaatactga cgccatcaca
     1321 atccacagca tcctggattg gattgaagac aacttggagt cgccgttgag tttagaaaaa
     1381 gttagtgaac gtagtggtta ctcaaagtgg caccttcagc gcatgtttaa gaaggaaacg
     1441 ggtcattcat tgggtcaata tattcgttct cgcaagatga ctgaaattgc ccagaaattg
     1501 aaagagtcta atgaacctat tttgtacctg gcggagcgtt acggctttga aagtcagcaa
     1561 acccttacac gtaccttcaa gaattacttt gacgttccac cacacaaata tcgtatgacc
     1621 aacatgcagg gtgagtcacg ttttttgcat ccgttgaatc attacaattc ctaataatcg
     1681 ctgggacgcc cgccatggtt cagccaaaaa acttaagacc gccggtcttg tccactacct
     1741 tgcagtaatg cggtggacag gatcggcggt tttcttttct cttctcaatt cttctgacct
     1801 gtaacgaata atagatagta aagtagtctc cgattgagtt ttctctgccg agtcccaccc
     1861 agttctgtga tttcagtaag ttggtaattg atacactgtt gcgagaactg ctgcctggta
     1921 gtagataggt tgttattgag taagaaggta aagtgaacga aatccctgaa actgagactg
     1981 tagaaaataa gcttcagcct gcggtccggt tgacagctag ctcagtccta ggtactgtgc
     2041 tagctcgctg ggacgcccgg ggactacact tacgaaacta ttgattgctc agcctgcggt
     2101 ccggccaaga gtacactagc tcagtcatcg ctgggacgcc cgggatccaa gagatttcta
     2161 cacgattgag cactgtctca gcctgcggtc cgggttcact gccgtatagg cagtaatttt
     2221 gtttaacttt aagaaggaga tatacatatg cgtaaaggcg aagaactgtt taccggtgtg
     2281 gttccgattc tggtggaact ggacggcgat gttaatggtc ataaattcag tgttcgcggc
     2341 gaaggtgaag gcgatgcgac gaacggcaaa ctgaccctga aatttatctg caccacgggt
     2401 aaactgccgg tcccgtggcc gacgctggtg accacgctga cctatggcgt tcaatgtttt
     2461 gcgcgttacc cggatcacat gaaacagcac gactttttca aatcggccat gccggaaggc
     2521 tatgtgcagg aacgtacgat tagctttaaa gacgatggta cgtataaaac ccgcgcggaa
     2581 gtgaaattcg aaggcgatac cctggttaac cgtatcgaac tgaaaggtat cgatttcaaa
     2641 gaagacggca atattctggg tcataaactg gaatataact tcaattccca caacgtgtac
     2701 atcaccgcgg ataaacagaa aaacggcatt aaagccaatt tcaaaatccg ccataatgtg
     2761 gaagatggta gcgttcagct ggccgaccac tatcagcaaa acacgccgat tggtgatggc
     2821 ccggtcctgc tgccggacaa tcactacctg agtacccagt ccgtgctgtc aaaagatccg
     2881 aacgaaaaac gtgaccacat ggtcctgctg gaatttgtga cggctgcggg tatcacccac
     2941 ggcatggacg aactgtataa aatgtcccgc cgtaatactg acgccatcac aatccacagc
     3001 atcctggatt ggattgaaga ctaataatcg ctgggacgcc cgcctgcagg ctcggtacca
     3061 aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc ctaatagata
     3121 aaggataggt ctggtagtgt tgttcgttct cgcaggtaaa tcaataatac tcagcagttc
     3181 cgtagacttt tcagtgggac agggtagcga taacagatag attgtaataa gacacagtag
     3241 gtgctcgtag ttgcgtgaag agaaccgctc aggaaatcca gtcagaagta ttggtaatcg
     3301 ttgaaaactc agtcgacgca cttactgaag acgtcctatt acactcgtcg ttggaaactg
     3361 aagatcagcc tgcggtccgg gttcactgcc gtataggcag taattttgtt taactttaag
     3421 aaggagatat acatatgaat cagtcattca tctcggacat cttatatgcc gacatcgaat
     3481 cgaaggctaa ggaacttaca gtcaattcca acaatactgt ccagccggtc gcgcttatgc
     3541 gcttaggagt tttcgttccc aaaccttcca agagcaaagg agaaagtaag gaaattgacg
     3601 ccaccaaagc cttctctcaa ctggagattg ctaaagcaga gggcatggtt agtaaaggag
     3661 aagaaaataa catggcactg attaaggaga acatgcacat gaagctgtac atggagggca
     3721 ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     3781 cccagaccat gagaatcaag gccgtcgagg gcggccctct ccccttcgcc ttcgacatcc
     3841 tggctaccag cttcatgtac ggcagcaaaa ccttcatcaa ccacacccag ggcatccccg
     3901 acttctttaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
     3961 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
     4021 acgtcaagat cagaggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac
     4081 tcggctggga ggcctccacc gagaccctgt accccgctga cggcggcctg gaaggcagag
     4141 ccgacatggc cctgaagctc gtgggcgggg gccacctgat ctgcaacttg aagaccacat
     4201 acagatccaa gaaacccgct aagaacctca agatgcccgg cgtctactat gtggacagaa
     4261 gactggaaag aatcaaggag gccgacaaag agacctacgt cgagcagcac gaggtggctg
     4321 tggccagata ctgcgacctc cctagcaaac tggggcacag agtctaataa tcgctgggac
     4381 gcccggcggc cgcggaaaca cagaaaaaag cccgcacctg acagtgcggg cttttttttt
     4441 cgaccaaagg tagcgaacga cgagtcactg ttgaggataa atactttctc tactaggcgc
     4501 ctgttacaca ggtcctcagc ggcgcgcctt tgtcggtgaa cgctctcctg agtaggacaa
     4561 atccgccggg agcggatttg aacgttgtga agcaacggcc cggagggtgg cgggcaggac
     4621 gcccgccata aactgccagg catcaaacta agcagaaggc catcctgacg gatggccttt
     4681 ttgcgtttca gatctaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     4741 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
     4801 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4861 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4921 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4981 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     5041 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     5101 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     5161 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     5221 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     5281 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     5341 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     5401 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     5461 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     5521 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     5581 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     5641 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     5701 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5761 tagaaaaata aacaaatagg catgctagcg cagaaacgtc ctagaagatg ccaggaggat
     5821 acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct ccgcccccct
     5881 gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa
     5941 agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc cgtcctgcgg
     6001 cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt gtagacagtt
     6061 cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg cgccttatcc
     6121 ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact ggcagcagcc
     6181 attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt ggcctaacag
     6241 aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag ttaccaggtt
     6301 aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg gttttttcgt
     6361 ttacagagca ggagattacg acgatcgtaa aaggatctca agaagatcct ttacggattc
     6421 ccgacaccat cactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     6481 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     6541 agctgactgg gttgctccta gggtctgatt cgttaccaat tatgacaact tgacggctac
     6601 atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt
     6661 tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac cgacggtggc
     6721 gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg
     6781 ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga
     6841 caagcaaaca tgctgtgcga cgctggcgat atcaaaattg ctgtctgcca ggtgatcgct
     6901 gatgtactga caagcctcgc gtacccgatt atccatcggt ggatggagcg actcgttaat
     6961 cgcttccatg cgccgcagta acaattgctc aagcagattt atcgccagca gctccgaata
     7021 gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac aggtcgctga aatgcggctg
     7081 gtgcgcttca tccgggcgaa agaaccccgt attggcaaat attgacggcc agttaagcca
     7141 ttcatgccag taggcgcgcg gacgaaagta aacccactgg tgataccatt cgcgagcctc
     7201 cggatgacga ccgtagtgat gaatctctcc tggcgggaac agcaaaatat cacccggtcg
     7261 gcaaacaaat tctcgtccct gatttttcac caccccctga ccgcgaatgg tgagattgag
     7321 aatataacct ttcattccca gcggtcggtc gataaaaaaa tcgagataac cgttggcctc
     7381 aatcggcgtt aaacccgcca ccagatgggc attaaacgag tatcccggca gcaggggatc
     7441 attttgcgct tcagccata
//
