LOCUS       sequence-305192-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate with sg-4t4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  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 7455)
  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_t4
FEATURES             Location/Qualifiers
     source          1..7455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(11..889)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        916..1200
                     /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     1123..1142
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1261..1280
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1383..1402
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    1469..1488
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1493..1515
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1523..2176
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2896..2930
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    3080..3099
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3104..3126
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3134..3847
                     /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    4267..4286
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             4291..4313
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      5540..5567
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5755..6570)
                     /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(5755..6570)
                     /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     6478..6497
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6571..6662)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6680..7315)
                     /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(6825..6844)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcgttaccaa ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac
       61 ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat
      121 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca
      181 gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct
      241 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga
      301 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat
      361 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct
      421 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga
      481 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg
      541 tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt
      601 aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc
      661 ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca
      721 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt
      781 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg
      841 cattaaacga gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac
      901 tcccgccatt cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg
      961 tcttttactg gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt
     1021 aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca
     1081 gaaaagtcca cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat
     1141 ccataagatt agcggttcct acctgacgct ttttatcgca actctctact gtttctccat
     1201 accgaattca taggatagat tctggaaact ttaccgtccg agctccagcc tgcggtccgg
     1261 gttcactgcc gtataggcag catcttagag tatgtagttt cagagctatg ctggaaacag
     1321 catagcaagt tgaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1381 gcgttcactg ccgtataggc agtcgctggg acgcccgctc gagcaataaa cagttgatag
     1441 ggcttctccg ttacagcctg cggtccgggt tcactgccgt ataggcagta attttgttta
     1501 actttaagaa ggagatatac atatggtttc ggttatcaaa ccagagatga aaatgcgtta
     1561 ctatatggat ggttcagtaa atggtcacga atttactatt gagggcgagg gtacgggacg
     1621 cccatacgag gggcaccagg aaatgacttt acgcgtcaca atggctgaag gcgggcctat
     1681 gccgtttgcg ttcgatcttg ttagtcatgt cttttgttac ggtcaccgtg tatttactaa
     1741 ataccccgag gaaattccag actatttcaa acaagccttc ccggaaggtt tgtcttggga
     1801 gcgcagttta gagtttgaag acggtggctc ggccagcgtg tcagctcata ttagtcttcg
     1861 cggcaataca ttttatcaca agtcaaagtt caccggcgtg aacttccccg cagacggccc
     1921 aatcatgcag aatcaaagtg ttgattggga accgtccaca gagaagatta cagcttccga
     1981 tggagtctta aagggcgatg taaccatgta cttaaaatta gaagggggag ggaaccataa
     2041 atgtcagatg aagactacct ataaggccgc aaaagagatt cttgaaatgc ccggagacca
     2101 ctacattggg catcgtttgg tccgtaagac agaaggaaat attactgaac aggtcgaaga
     2161 cgctgtggca cacagcatgt cccgccgtaa tactgacgcc atcacaatcc acagcatcct
     2221 ggattggatt gaagacaact tggagtcgcc gttgagttta gaaaaagtta gtgaacgtag
     2281 tggttactca aagtggcacc ttcagcgcat gtttaagaag gaaacgggtc attcattggg
     2341 tcaatatatt cgttctcgca agatgactga aattgcccag aaattgaaag agtctaatga
     2401 acctattttg tacctggcgg agcgttacgg ctttgaaagt cagcaaaccc ttacacgtac
     2461 cttcaagaat tactttgacg ttccaccaca caaatatcgt atgaccaaca tgcagggtga
     2521 gtcacgtttt ttgcatccgt tgaatcatta caattcctaa taatcgctgg gacgcccgcc
     2581 atggttcagc caaaaaactt aagaccgccg gtcttgtcca ctaccttgca gtaatgcggt
     2641 ggacaggatc ggcggttttc ttttctcttc tcaattcttc tgacctgtaa cgaataatag
     2701 atagtaaagt agtctccgat tgagttttct ctgccgagtc ccacccagtt ctgtgatttc
     2761 agtaagttgg taattgatac actgttgcga gaactgctgc ctggtagtag ataggttgtt
     2821 attgagtaag aaggtaaagt gaacgaaatc cctgaaactg agactgtaga aaataagctt
     2881 cagcctgcgg tccggttgac agctagctca gtcctaggta ctgtgctagc tcgctgggac
     2941 gcccggggac tacacttacg aaactattga ttgctcagcc tgcggtccgg ccatacatac
     3001 tctaagatgt gtctcgctgg gacgcccggg atccaagaga tttctacacg attgagcact
     3061 gtctcagcct gcggtccggg ttcactgccg tataggcagt aattttgttt aactttaaga
     3121 aggagatata catatgcgta aaggcgaaga actgtttacc ggtgtggttc cgattctggt
     3181 ggaactggac ggcgatgtta atggtcataa attcagtgtt cgcggcgaag gtgaaggcga
     3241 tgcgacgaac ggcaaactga ccctgaaatt tatctgcacc acgggtaaac tgccggtccc
     3301 gtggccgacg ctggtgacca cgctgaccta tggcgttcaa tgttttgcgc gttacccgga
     3361 tcacatgaaa cagcacgact ttttcaaatc ggccatgccg gaaggctatg tgcaggaacg
     3421 tacgattagc tttaaagacg atggtacgta taaaacccgc gcggaagtga aattcgaagg
     3481 cgataccctg gttaaccgta tcgaactgaa aggtatcgat ttcaaagaag acggcaatat
     3541 tctgggtcat aaactggaat ataacttcaa ttcccacaac gtgtacatca ccgcggataa
     3601 acagaaaaac ggcattaaag ccaatttcaa aatccgccat aatgtggaag atggtagcgt
     3661 tcagctggcc gaccactatc agcaaaacac gccgattggt gatggcccgg tcctgctgcc
     3721 ggacaatcac tacctgagta cccagtccgt gctgtcaaaa gatccgaacg aaaaacgtga
     3781 ccacatggtc ctgctggaat ttgtgacggc tgcgggtatc acccacggca tggacgaact
     3841 gtataaaatg tcccgccgta atactgacgc catcacaatc cacagcatcc tggattggat
     3901 tgaagactaa taatcgctgg gacgcccgcc tgcaggctcg gtaccaaatt ccagaaaaga
     3961 ggcctcccga aaggggggcc ttttttcgtt ttggtcctaa tagataaagg ataggtctgg
     4021 tagtgttgtt cgttctcgca ggtaaatcaa taatactcag cagttccgta gacttttcag
     4081 tgggacaggg tagcgataac agatagattg taataagaca cagtaggtgc tcgtagttgc
     4141 gtgaagagaa ccgctcagga aatccagtca gaagtattgg taatcgttga aaactcagtc
     4201 gacgcactta ctgaagacgt cctattacac tcgtcgttgg aaactgaaga tcagcctgcg
     4261 gtccgggttc actgccgtat aggcagtaat tttgtttaac tttaagaagg agatatacat
     4321 atgaatcagt cattcatctc ggacatctta tatgccgaca tcgaatcgaa ggctaaggaa
     4381 cttacagtca attccaacaa tactgtccag ccggtcgcgc ttatgcgctt aggagttttc
     4441 gttcccaaac cttccaagag caaaggagaa agtaaggaaa ttgacgccac caaagccttc
     4501 tctcaactgg agattgctaa agcagagggc atggttagta aaggagaaga aaataacatg
     4561 gcactgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaaccac
     4621 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     4681 atcaaggccg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc
     4741 atgtacggca gcaaaacctt catcaaccac acccagggca tccccgactt ctttaagcag
     4801 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     4861 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     4921 ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     4981 tccaccgaga ccctgtaccc cgctgacggc ggcctggaag gcagagccga catggccctg
     5041 aagctcgtgg gcgggggcca cctgatctgc aacttgaaga ccacatacag atccaagaaa
     5101 cccgctaaga acctcaagat gcccggcgtc tactatgtgg acagaagact ggaaagaatc
     5161 aaggaggccg acaaagagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc
     5221 gacctcccta gcaaactggg gcacagagtc taataatcgc tgggacgccc ggcggccgcg
     5281 gaaacacaga aaaaagcccg cacctgacag tgcgggcttt ttttttcgac caaaggtagc
     5341 gaacgacgag tcactgttga ggataaatac tttctctact aggcgcctgt tacacaggtc
     5401 ctcagcggcg cgcctttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     5461 gatttgaacg ttgtgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     5521 gccaggcatc aaactaagca gaaggccatc ctgacggatg gcctttttgc gtttcagatc
     5581 taccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga
     5641 caagctgacg accgggtctc cgcaagtggc acttttcggg gaaatgtgcg cggaacccct
     5701 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     5761 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     5821 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     5881 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     5941 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     6001 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     6061 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     6121 agcgagacga aatacgcggt cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     6181 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     6241 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     6301 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     6361 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     6421 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     6481 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     6541 gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat
     6601 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6661 aataggcatg ctagcgcaga aacgtcctag aagatgccag gaggatactt agcagagaga
     6721 caataaggcc ggagcgaagc cgtttttcca taggctccgc ccccctgacg aacatcacga
     6781 aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6841 tccccctgat ggctccctct tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg
     6901 tggaggcttt acccaaatca ccacgtcccg ttccgtgtag acagttcgct ccaagctggg
     6961 ctgtgtgcaa gaaccccccg ttcagcccga ctgctgcgcc ttatccggta actatcatct
     7021 tgagtccaac ccggaaagac acgacaaaac gccactggca gcagccattg gtaactgaga
     7081 attagtggat ttagatatcg agagtcttga agtggtggcc taacagaggc tacactgaaa
     7141 ggacagtatt tggtatctgc gctccactaa agccagttac caggttaagc agttccccaa
     7201 ctgacttaac cttcgatcaa accgcctccc caggcggttt tttcgtttac agagcaggag
     7261 attacgacga tcgtaaaagg atctcaagaa gatcctttac ggattcccga caccatcact
     7321 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     7381 taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     7441 ctcctagggt ctgat
//
