LOCUS       sequence-305191-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NOT gate with sg-1t4.
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: pJ2044_t4
FEATURES             Location/Qualifiers
     source          1..7455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..320
                     /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     243..262
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    381..400
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    503..522
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    589..608
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             613..635
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             643..1296
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2016..2050
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2200..2219
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2224..2246
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2254..2967
                     /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    3387..3406
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3411..3433
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      4660..4687
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4875..5690)
                     /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(4875..5690)
                     /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     5598..5617
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5691..5782)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5800..6435)
                     /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(5945..5964)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6586..7455,1..9))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc catattgcat
       61 cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac cggtaacccc
      121 gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa cgcgtaacaa
      181 aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg tcacactttg
      241 ctatgccata gcatttttat ccataagatt agcggttcct acctgacgct ttttatcgca
      301 actctctact gtttctccat accgaattca taggatagat tctggaaact ttaccgtccg
      361 agctccagcc tgcggtccgg gttcactgcc gtataggcag aagctagact ctagtggttt
      421 cagagctatg ctggaaacag catagcaagt tgaaataagg ctagtccgtt atcaacttga
      481 aaaagtggca ccgagtcggt gcgttcactg ccgtataggc agtcgctggg acgcccgctc
      541 gagcaataaa cagttgatag ggcttctccg ttacagcctg cggtccgggt tcactgccgt
      601 ataggcagta attttgttta actttaagaa ggagatatac atatggtttc ggttatcaaa
      661 ccagagatga aaatgcgtta ctatatggat ggttcagtaa atggtcacga atttactatt
      721 gagggcgagg gtacgggacg cccatacgag gggcaccagg aaatgacttt acgcgtcaca
      781 atggctgaag gcgggcctat gccgtttgcg ttcgatcttg ttagtcatgt cttttgttac
      841 ggtcaccgtg tatttactaa ataccccgag gaaattccag actatttcaa acaagccttc
      901 ccggaaggtt tgtcttggga gcgcagttta gagtttgaag acggtggctc ggccagcgtg
      961 tcagctcata ttagtcttcg cggcaataca ttttatcaca agtcaaagtt caccggcgtg
     1021 aacttccccg cagacggccc aatcatgcag aatcaaagtg ttgattggga accgtccaca
     1081 gagaagatta cagcttccga tggagtctta aagggcgatg taaccatgta cttaaaatta
     1141 gaagggggag ggaaccataa atgtcagatg aagactacct ataaggccgc aaaagagatt
     1201 cttgaaatgc ccggagacca ctacattggg catcgtttgg tccgtaagac agaaggaaat
     1261 attactgaac aggtcgaaga cgctgtggca cacagcatgt cccgccgtaa tactgacgcc
     1321 atcacaatcc acagcatcct ggattggatt gaagacaact tggagtcgcc gttgagttta
     1381 gaaaaagtta gtgaacgtag tggttactca aagtggcacc ttcagcgcat gtttaagaag
     1441 gaaacgggtc attcattggg tcaatatatt cgttctcgca agatgactga aattgcccag
     1501 aaattgaaag agtctaatga acctattttg tacctggcgg agcgttacgg ctttgaaagt
     1561 cagcaaaccc ttacacgtac cttcaagaat tactttgacg ttccaccaca caaatatcgt
     1621 atgaccaaca tgcagggtga gtcacgtttt ttgcatccgt tgaatcatta caattcctaa
     1681 taatcgctgg gacgcccgcc atggttcagc caaaaaactt aagaccgccg gtcttgtcca
     1741 ctaccttgca gtaatgcggt ggacaggatc ggcggttttc ttttctcttc tcaattcttc
     1801 tgacctgtaa cgaataatag atagtaaagt agtctccgat tgagttttct ctgccgagtc
     1861 ccacccagtt ctgtgatttc agtaagttgg taattgatac actgttgcga gaactgctgc
     1921 ctggtagtag ataggttgtt attgagtaag aaggtaaagt gaacgaaatc cctgaaactg
     1981 agactgtaga aaataagctt cagcctgcgg tccggttgac agctagctca gtcctaggta
     2041 ctgtgctagc tcgctgggac gcccggggac tacacttacg aaactattga ttgctcagcc
     2101 tgcggtccgg ccacactaga gtctagcttg agatcgctgg gacgcccggg atccaagaga
     2161 tttctacacg attgagcact gtctcagcct gcggtccggg ttcactgccg tataggcagt
     2221 aattttgttt aactttaaga aggagatata catatgcgta aaggcgaaga actgtttacc
     2281 ggtgtggttc cgattctggt ggaactggac ggcgatgtta atggtcataa attcagtgtt
     2341 cgcggcgaag gtgaaggcga tgcgacgaac ggcaaactga ccctgaaatt tatctgcacc
     2401 acgggtaaac tgccggtccc gtggccgacg ctggtgacca cgctgaccta tggcgttcaa
     2461 tgttttgcgc gttacccgga tcacatgaaa cagcacgact ttttcaaatc ggccatgccg
     2521 gaaggctatg tgcaggaacg tacgattagc tttaaagacg atggtacgta taaaacccgc
     2581 gcggaagtga aattcgaagg cgataccctg gttaaccgta tcgaactgaa aggtatcgat
     2641 ttcaaagaag acggcaatat tctgggtcat aaactggaat ataacttcaa ttcccacaac
     2701 gtgtacatca ccgcggataa acagaaaaac ggcattaaag ccaatttcaa aatccgccat
     2761 aatgtggaag atggtagcgt tcagctggcc gaccactatc agcaaaacac gccgattggt
     2821 gatggcccgg tcctgctgcc ggacaatcac tacctgagta cccagtccgt gctgtcaaaa
     2881 gatccgaacg aaaaacgtga ccacatggtc ctgctggaat ttgtgacggc tgcgggtatc
     2941 acccacggca tggacgaact gtataaaatg tcccgccgta atactgacgc catcacaatc
     3001 cacagcatcc tggattggat tgaagactaa taatcgctgg gacgcccgcc tgcaggctcg
     3061 gtaccaaatt ccagaaaaga ggcctcccga aaggggggcc ttttttcgtt ttggtcctaa
     3121 tagataaagg ataggtctgg tagtgttgtt cgttctcgca ggtaaatcaa taatactcag
     3181 cagttccgta gacttttcag tgggacaggg tagcgataac agatagattg taataagaca
     3241 cagtaggtgc tcgtagttgc gtgaagagaa ccgctcagga aatccagtca gaagtattgg
     3301 taatcgttga aaactcagtc gacgcactta ctgaagacgt cctattacac tcgtcgttgg
     3361 aaactgaaga tcagcctgcg gtccgggttc actgccgtat aggcagtaat tttgtttaac
     3421 tttaagaagg agatatacat atgaatcagt cattcatctc ggacatctta tatgccgaca
     3481 tcgaatcgaa ggctaaggaa cttacagtca attccaacaa tactgtccag ccggtcgcgc
     3541 ttatgcgctt aggagttttc gttcccaaac cttccaagag caaaggagaa agtaaggaaa
     3601 ttgacgccac caaagccttc tctcaactgg agattgctaa agcagagggc atggttagta
     3661 aaggagaaga aaataacatg gcactgatta aggagaacat gcacatgaag ctgtacatgg
     3721 agggcaccgt gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg
     3781 agggcaccca gaccatgaga atcaaggccg tcgagggcgg ccctctcccc ttcgccttcg
     3841 acatcctggc taccagcttc atgtacggca gcaaaacctt catcaaccac acccagggca
     3901 tccccgactt ctttaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     3961 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     4021 tctacaacgt caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga
     4081 aaacactcgg ctgggaggcc tccaccgaga ccctgtaccc cgctgacggc ggcctggaag
     4141 gcagagccga catggccctg aagctcgtgg gcgggggcca cctgatctgc aacttgaaga
     4201 ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg
     4261 acagaagact ggaaagaatc aaggaggccg acaaagagac ctacgtcgag cagcacgagg
     4321 tggctgtggc cagatactgc gacctcccta gcaaactggg gcacagagtc taataatcgc
     4381 tgggacgccc ggcggccgcg gaaacacaga aaaaagcccg cacctgacag tgcgggcttt
     4441 ttttttcgac caaaggtagc gaacgacgag tcactgttga ggataaatac tttctctact
     4501 aggcgcctgt tacacaggtc ctcagcggcg cgcctttgtc ggtgaacgct ctcctgagta
     4561 ggacaaatcc gccgggagcg gatttgaacg ttgtgaagca acggcccgga gggtggcggg
     4621 caggacgccc gccataaact gccaggcatc aaactaagca gaaggccatc ctgacggatg
     4681 gcctttttgc gtttcagatc taccggtaaa ccagcaatag acataagcgg ctatttaacg
     4741 accctgccct gaaccgacga caagctgacg accgggtctc cgcaagtggc acttttcggg
     4801 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4861 tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     4921 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     4981 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     5041 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     5101 ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact
     5161 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     5221 ttcattcgtg attgcgcctg agcgagacga aatacgcggt cgctgttaaa aggacaatta
     5281 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     5341 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     5401 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     5461 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     5521 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     5581 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     5641 gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt
     5701 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5761 tgtatttaga aaaataaaca aataggcatg ctagcgcaga aacgtcctag aagatgccag
     5821 gaggatactt agcagagaga caataaggcc ggagcgaagc cgtttttcca taggctccgc
     5881 ccccctgacg aacatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga
     5941 ctataaagat accaggcgtt tccccctgat ggctccctct tgcgctctcc tgttcccgtc
     6001 ctgcggcgtc cgtgttgtgg tggaggcttt acccaaatca ccacgtcccg ttccgtgtag
     6061 acagttcgct ccaagctggg ctgtgtgcaa gaaccccccg ttcagcccga ctgctgcgcc
     6121 ttatccggta actatcatct tgagtccaac ccggaaagac acgacaaaac gccactggca
     6181 gcagccattg gtaactgaga attagtggat ttagatatcg agagtcttga agtggtggcc
     6241 taacagaggc tacactgaaa ggacagtatt tggtatctgc gctccactaa agccagttac
     6301 caggttaagc agttccccaa ctgacttaac cttcgatcaa accgcctccc caggcggttt
     6361 tttcgtttac agagcaggag attacgacga tcgtaaaagg atctcaagaa gatcctttac
     6421 ggattcccga caccatcact ctagatttca gtgcaattta tctcttcaaa tgtagcacct
     6481 gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc
     6541 ggaaggagct gactgggttg ctcctagggt ctgattcgtt accaattatg acaacttgac
     6601 ggctacatca ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt
     6661 gcatttttta aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac
     6721 ggtggcgata ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc
     6781 ctcgcgccag cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga
     6841 cggcgacaag caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg
     6901 atcgctgatg tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc
     6961 gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc
     7021 cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg
     7081 cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt
     7141 aagccattca tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg
     7201 agcctccgga tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc
     7261 cggtcggcaa acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag
     7321 attgagaata taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt
     7381 ggcctcaatc ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag
     7441 gggatcattt tgcgc
//
