LOCUS       sequence-273321-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control ("C") lacking sg-1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7295)
  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 7295)
  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_N2only
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        27..311
                     /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     234..253
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    420..439
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             444..466
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             474..1127
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        1847..1881
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2031..2050
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2055..2077
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2085..2798
                     /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    3218..3237
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             3242..3264
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      4491..4518
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(4706..5521)
                     /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(4706..5521)
                     /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     5429..5448
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5522..5613)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5631..6266)
                     /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(5776..5795)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6417..7295)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg
       61 ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa
      121 aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta
      181 taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat
      241 agcattttta tccataagat tagcggttcc tacctgacgc tttttatcgc aactctctac
      301 tgtttctcca taccgaattc ataggataga ttctggaaac tttaccgtcc gagctcaggc
      361 ttaccttact cgagcaataa acagttgata gggcttctcc gttacagcct gcggtccggg
      421 ttcactgccg tataggcagt aattttgttt aactttaaga aggagatata catatggttt
      481 cggttatcaa accagagatg aaaatgcgtt actatatgga tggttcagta aatggtcacg
      541 aatttactat tgagggcgag ggtacgggac gcccatacga ggggcaccag gaaatgactt
      601 tacgcgtcac aatggctgaa ggcgggccta tgccgtttgc gttcgatctt gttagtcatg
      661 tcttttgtta cggtcaccgt gtatttacta aataccccga ggaaattcca gactatttca
      721 aacaagcctt cccggaaggt ttgtcttggg agcgcagttt agagtttgaa gacggtggct
      781 cggccagcgt gtcagctcat attagtcttc gcggcaatac attttatcac aagtcaaagt
      841 tcaccggcgt gaacttcccc gcagacggcc caatcatgca gaatcaaagt gttgattggg
      901 aaccgtccac agagaagatt acagcttccg atggagtctt aaagggcgat gtaaccatgt
      961 acttaaaatt agaaggggga gggaaccata aatgtcagat gaagactacc tataaggccg
     1021 caaaagagat tcttgaaatg cccggagacc actacattgg gcatcgtttg gtccgtaaga
     1081 cagaaggaaa tattactgaa caggtcgaag acgctgtggc acacagcatg tcccgccgta
     1141 atactgacgc catcacaatc cacagcatcc tggattggat tgaagacaac ttggagtcgc
     1201 cgttgagttt agaaaaagtt agtgaacgta gtggttactc aaagtggcac cttcagcgca
     1261 tgtttaagaa ggaaacgggt cattcattgg gtcaatatat tcgttctcgc aagatgactg
     1321 aaattgccca gaaattgaaa gagtctaatg aacctatttt gtacctggcg gagcgttacg
     1381 gctttgaaag tcagcaaacc cttacacgta ccttcaagaa ttactttgac gttccaccac
     1441 acaaatatcg tatgaccaac atgcagggtg agtcacgttt tttgcatccg ttgaatcatt
     1501 acaattccta ataatcgctg ggacgcccgc catggttcag ccaaaaaact taagaccgcc
     1561 ggtcttgtcc actaccttgc agtaatgcgg tggacaggat cggcggtttt cttttctctt
     1621 ctcaattctt ctgacctgta acgaataata gatagtaaag tagtctccga ttgagttttc
     1681 tctgccgagt cccacccagt tctgtgattt cagtaagttg gtaattgata cactgttgcg
     1741 agaactgctg cctggtagta gataggttgt tattgagtaa gaaggtaaag tgaacgaaat
     1801 ccctgaaact gagactgtag aaaataagct tcagcctgcg gtccggttga cagctagctc
     1861 agtcctaggt actgtgctag ctcgctggga cgcccgggga ctacacttac gaaactattg
     1921 attgctcagc ctgcggtccg gccacactag agtctagctt gagatcgctg ggacgcccgg
     1981 gatccaagag atttctacac gattgagcac tgtctcagcc tgcggtccgg gttcactgcc
     2041 gtataggcag taattttgtt taactttaag aaggagatat acatatgcgt aaaggcgaag
     2101 aactgtttac cggtgtggtt ccgattctgg tggaactgga cggcgatgtt aatggtcata
     2161 aattcagtgt tcgcggcgaa ggtgaaggcg atgcgacgaa cggcaaactg accctgaaat
     2221 ttatctgcac cacgggtaaa ctgccggtcc cgtggccgac gctggtgacc acgctgacct
     2281 atggcgttca atgttttgcg cgttacccgg atcacatgaa acagcacgac tttttcaaat
     2341 cggccatgcc ggaaggctat gtgcaggaac gtacgattag ctttaaagac gatggtacgt
     2401 ataaaacccg cgcggaagtg aaattcgaag gcgataccct ggttaaccgt atcgaactga
     2461 aaggtatcga tttcaaagaa gacggcaata ttctgggtca taaactggaa tataacttca
     2521 attcccacaa cgtgtacatc accgcggata aacagaaaaa cggcattaaa gccaatttca
     2581 aaatccgcca taatgtggaa gatggtagcg ttcagctggc cgaccactat cagcaaaaca
     2641 cgccgattgg tgatggcccg gtcctgctgc cggacaatca ctacctgagt acccagtccg
     2701 tgctgtcaaa agatccgaac gaaaaacgtg accacatggt cctgctggaa tttgtgacgg
     2761 ctgcgggtat cacccacggc atggacgaac tgtataaaat gtcccgccgt aatactgacg
     2821 ccatcacaat ccacagcatc ctggattgga ttgaagacta ataatcgctg ggacgcccgc
     2881 ctgcaggctc ggtaccaaat tccagaaaag aggcctcccg aaaggggggc cttttttcgt
     2941 tttggtccta atagataaag gataggtctg gtagtgttgt tcgttctcgc aggtaaatca
     3001 ataatactca gcagttccgt agacttttca gtgggacagg gtagcgataa cagatagatt
     3061 gtaataagac acagtaggtg ctcgtagttg cgtgaagaga accgctcagg aaatccagtc
     3121 agaagtattg gtaatcgttg aaaactcagt cgacgcactt actgaagacg tcctattaca
     3181 ctcgtcgttg gaaactgaag atcagcctgc ggtccgggtt cactgccgta taggcagtaa
     3241 ttttgtttaa ctttaagaag gagatataca tatgaatcag tcattcatct cggacatctt
     3301 atatgccgac atcgaatcga aggctaagga acttacagtc aattccaaca atactgtcca
     3361 gccggtcgcg cttatgcgct taggagtttt cgttcccaaa ccttccaaga gcaaaggaga
     3421 aagtaaggaa attgacgcca ccaaagcctt ctctcaactg gagattgcta aagcagaggg
     3481 catggttagt aaaggagaag aaaataacat ggcactgatt aaggagaaca tgcacatgaa
     3541 gctgtacatg gagggcaccg tgaacaacca ccacttcaag tgcacatccg agggcgaagg
     3601 caagccctac gagggcaccc agaccatgag aatcaaggcc gtcgagggcg gccctctccc
     3661 cttcgccttc gacatcctgg ctaccagctt catgtacggc agcaaaacct tcatcaacca
     3721 cacccagggc atccccgact tctttaagca gtccttccct gagggcttca catgggagag
     3781 agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca gcctccagga
     3841 cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcccatcca acggccctgt
     3901 gatgcagaag aaaacactcg gctgggaggc ctccaccgag accctgtacc ccgctgacgg
     3961 cggcctggaa ggcagagccg acatggccct gaagctcgtg ggcgggggcc acctgatctg
     4021 caacttgaag accacataca gatccaagaa acccgctaag aacctcaaga tgcccggcgt
     4081 ctactatgtg gacagaagac tggaaagaat caaggaggcc gacaaagaga cctacgtcga
     4141 gcagcacgag gtggctgtgg ccagatactg cgacctccct agcaaactgg ggcacagagt
     4201 ctaataatcg ctgggacgcc cggcggccgc ggaaacacag aaaaaagccc gcacctgaca
     4261 gtgcgggctt tttttttcga ccaaaggtag cgaacgacga gtcactgttg aggataaata
     4321 ctttctctac taggcgcctg ttacacaggt cctcagcggc gcgcctttgt cggtgaacgc
     4381 tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg
     4441 agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat
     4501 cctgacggat ggcctttttg cgtttcagat ctaccggtaa accagcaata gacataagcg
     4561 gctatttaac gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg
     4621 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4681 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     4741 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     4801 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     4861 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     4921 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     4981 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     5041 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa
     5101 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     5161 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     5221 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     5281 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     5341 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     5401 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     5461 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     5521 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5581 acatatttga atgtatttag aaaaataaac aaataggcat gctagcgcag aaacgtccta
     5641 gaagatgcca ggaggatact tagcagagag acaataaggc cggagcgaag ccgtttttcc
     5701 ataggctccg cccccctgac gaacatcacg aaatctgacg ctcaaatcag tggtggcgaa
     5761 acccgacagg actataaaga taccaggcgt ttccccctga tggctccctc ttgcgctctc
     5821 ctgttcccgt cctgcggcgt ccgtgttgtg gtggaggctt tacccaaatc accacgtccc
     5881 gttccgtgta gacagttcgc tccaagctgg gctgtgtgca agaacccccc gttcagcccg
     5941 actgctgcgc cttatccggt aactatcatc ttgagtccaa cccggaaaga cacgacaaaa
     6001 cgccactggc agcagccatt ggtaactgag aattagtgga tttagatatc gagagtcttg
     6061 aagtggtggc ctaacagagg ctacactgaa aggacagtat ttggtatctg cgctccacta
     6121 aagccagtta ccaggttaag cagttcccca actgacttaa ccttcgatca aaccgcctcc
     6181 ccaggcggtt ttttcgttta cagagcagga gattacgacg atcgtaaaag gatctcaaga
     6241 agatccttta cggattcccg acaccatcac tctagatttc agtgcaattt atctcttcaa
     6301 atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc
     6361 ccgcgcccac cggaaggagc tgactgggtt gctcctaggg tctgattcgt taccaattat
     6421 gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg
     6481 ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca
     6541 ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg
     6601 atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt
     6661 gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg
     6721 tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga
     6781 tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc
     6841 gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg
     6901 tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt
     6961 gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga
     7021 taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc
     7081 aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg
     7141 cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg
     7201 agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt aaacgagtat
     7261 cccggcagca ggggatcatt ttgcgcttca gccat
//
