LOCUS       sequence-305193-        7295 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Control ("C") lacking sg-2.
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: pJ2043_N2only
FEATURES             Location/Qualifiers
     source          1..7295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        864..1148
                     /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     1071..1090
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    1257..1276
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1281..1303
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1311..1964
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     promoter        2684..2718
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    2868..2887
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2892..2914
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2922..3635
                     /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    4055..4074
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             4079..4101
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     terminator      5328..5355
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(5543..6358)
                     /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(5543..6358)
                     /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     6266..6285
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(6359..6450)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6468..7103)
                     /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(6613..6632)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7254..7295,1..837))
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
ORIGIN
        1 accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata
       61 gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct
      121 caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc
      181 taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac
      241 gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg
      301 tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa
      361 caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc
      421 gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa
      481 gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg
      541 acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg
      601 aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg
      661 atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag
      721 cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac
      781 cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact
      841 tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg
      901 tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag
      961 cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa
     1021 tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc
     1081 atttttatcc ataagattag cggttcctac ctgacgcttt ttatcgcaac tctctactgt
     1141 ttctccatac cgaattcata ggatagattc tggaaacttt accgtccgag ctcaggctta
     1201 ccttactcga gcaataaaca gttgataggg cttctccgtt acagcctgcg gtccgggttc
     1261 actgccgtat aggcagtaat tttgtttaac tttaagaagg agatatacat atggtttcgg
     1321 ttatcaaacc agagatgaaa atgcgttact atatggatgg ttcagtaaat ggtcacgaat
     1381 ttactattga gggcgagggt acgggacgcc catacgaggg gcaccaggaa atgactttac
     1441 gcgtcacaat ggctgaaggc gggcctatgc cgtttgcgtt cgatcttgtt agtcatgtct
     1501 tttgttacgg tcaccgtgta tttactaaat accccgagga aattccagac tatttcaaac
     1561 aagccttccc ggaaggtttg tcttgggagc gcagtttaga gtttgaagac ggtggctcgg
     1621 ccagcgtgtc agctcatatt agtcttcgcg gcaatacatt ttatcacaag tcaaagttca
     1681 ccggcgtgaa cttccccgca gacggcccaa tcatgcagaa tcaaagtgtt gattgggaac
     1741 cgtccacaga gaagattaca gcttccgatg gagtcttaaa gggcgatgta accatgtact
     1801 taaaattaga agggggaggg aaccataaat gtcagatgaa gactacctat aaggccgcaa
     1861 aagagattct tgaaatgccc ggagaccact acattgggca tcgtttggtc cgtaagacag
     1921 aaggaaatat tactgaacag gtcgaagacg ctgtggcaca cagcatgtcc cgccgtaata
     1981 ctgacgccat cacaatccac agcatcctgg attggattga agacaacttg gagtcgccgt
     2041 tgagtttaga aaaagttagt gaacgtagtg gttactcaaa gtggcacctt cagcgcatgt
     2101 ttaagaagga aacgggtcat tcattgggtc aatatattcg ttctcgcaag atgactgaaa
     2161 ttgcccagaa attgaaagag tctaatgaac ctattttgta cctggcggag cgttacggct
     2221 ttgaaagtca gcaaaccctt acacgtacct tcaagaatta ctttgacgtt ccaccacaca
     2281 aatatcgtat gaccaacatg cagggtgagt cacgtttttt gcatccgttg aatcattaca
     2341 attcctaata atcgctggga cgcccgccat ggttcagcca aaaaacttaa gaccgccggt
     2401 cttgtccact accttgcagt aatgcggtgg acaggatcgg cggttttctt ttctcttctc
     2461 aattcttctg acctgtaacg aataatagat agtaaagtag tctccgattg agttttctct
     2521 gccgagtccc acccagttct gtgatttcag taagttggta attgatacac tgttgcgaga
     2581 actgctgcct ggtagtagat aggttgttat tgagtaagaa ggtaaagtga acgaaatccc
     2641 tgaaactgag actgtagaaa ataagcttca gcctgcggtc cggttgacag ctagctcagt
     2701 cctaggtact gtgctagctc gctgggacgc ccggggacta cacttacgaa actattgatt
     2761 gctcagcctg cggtccggcc aagtacttag tacacactga ttcgctggga cgcccgggat
     2821 ccaagagatt tctacacgat tgagcactgt ctcagcctgc ggtccgggtt cactgccgta
     2881 taggcagtaa ttttgtttaa ctttaagaag gagatataca tatgcgtaaa ggcgaagaac
     2941 tgtttaccgg tgtggttccg attctggtgg aactggacgg cgatgttaat ggtcataaat
     3001 tcagtgttcg cggcgaaggt gaaggcgatg cgacgaacgg caaactgacc ctgaaattta
     3061 tctgcaccac gggtaaactg ccggtcccgt ggccgacgct ggtgaccacg ctgacctatg
     3121 gcgttcaatg ttttgcgcgt tacccggatc acatgaaaca gcacgacttt ttcaaatcgg
     3181 ccatgccgga aggctatgtg caggaacgta cgattagctt taaagacgat ggtacgtata
     3241 aaacccgcgc ggaagtgaaa ttcgaaggcg ataccctggt taaccgtatc gaactgaaag
     3301 gtatcgattt caaagaagac ggcaatattc tgggtcataa actggaatat aacttcaatt
     3361 cccacaacgt gtacatcacc gcggataaac agaaaaacgg cattaaagcc aatttcaaaa
     3421 tccgccataa tgtggaagat ggtagcgttc agctggccga ccactatcag caaaacacgc
     3481 cgattggtga tggcccggtc ctgctgccgg acaatcacta cctgagtacc cagtccgtgc
     3541 tgtcaaaaga tccgaacgaa aaacgtgacc acatggtcct gctggaattt gtgacggctg
     3601 cgggtatcac ccacggcatg gacgaactgt ataaaatgtc ccgccgtaat actgacgcca
     3661 tcacaatcca cagcatcctg gattggattg aagactaata atcgctggga cgcccgcctg
     3721 caggctcggt accaaattcc agaaaagagg cctcccgaaa ggggggcctt ttttcgtttt
     3781 ggtcctaata gataaaggat aggtctggta gtgttgttcg ttctcgcagg taaatcaata
     3841 atactcagca gttccgtaga cttttcagtg ggacagggta gcgataacag atagattgta
     3901 ataagacaca gtaggtgctc gtagttgcgt gaagagaacc gctcaggaaa tccagtcaga
     3961 agtattggta atcgttgaaa actcagtcga cgcacttact gaagacgtcc tattacactc
     4021 gtcgttggaa actgaagatc agcctgcggt ccgggttcac tgccgtatag gcagtaattt
     4081 tgtttaactt taagaaggag atatacatat gaatcagtca ttcatctcgg acatcttata
     4141 tgccgacatc gaatcgaagg ctaaggaact tacagtcaat tccaacaata ctgtccagcc
     4201 ggtcgcgctt atgcgcttag gagttttcgt tcccaaacct tccaagagca aaggagaaag
     4261 taaggaaatt gacgccacca aagccttctc tcaactggag attgctaaag cagagggcat
     4321 ggttagtaaa ggagaagaaa ataacatggc actgattaag gagaacatgc acatgaagct
     4381 gtacatggag ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa
     4441 gccctacgag ggcacccaga ccatgagaat caaggccgtc gagggcggcc ctctcccctt
     4501 cgccttcgac atcctggcta ccagcttcat gtacggcagc aaaaccttca tcaaccacac
     4561 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt
     4621 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     4681 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc ccatccaacg gccctgtgat
     4741 gcagaagaaa acactcggct gggaggcctc caccgagacc ctgtaccccg ctgacggcgg
     4801 cctggaaggc agagccgaca tggccctgaa gctcgtgggc gggggccacc tgatctgcaa
     4861 cttgaagacc acatacagat ccaagaaacc cgctaagaac ctcaagatgc ccggcgtcta
     4921 ctatgtggac agaagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
     4981 gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc acagagtcta
     5041 ataatcgctg ggacgcccgg cggccgcgga aacacagaaa aaagcccgca cctgacagtg
     5101 cgggcttttt ttttcgacca aaggtagcga acgacgagtc actgttgagg ataaatactt
     5161 tctctactag gcgcctgtta cacaggtcct cagcggcgcg cctttgtcgg tgaacgctct
     5221 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg
     5281 gtggcgggca ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatcct
     5341 gacggatggc ctttttgcgt ttcagatcta ccggtaaacc agcaatagac ataagcggct
     5401 atttaacgac cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac
     5461 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5521 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     5581 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     5641 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     5701 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     5761 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     5821 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     5881 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag
     5941 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     6001 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     6061 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     6121 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     6181 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     6241 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     6301 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac
     6361 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6421 tatttgaatg tatttagaaa aataaacaaa taggcatgct agcgcagaaa cgtcctagaa
     6481 gatgccagga ggatacttag cagagagaca ataaggccgg agcgaagccg tttttccata
     6541 ggctccgccc ccctgacgaa catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     6601 cgacaggact ataaagatac caggcgtttc cccctgatgg ctccctcttg cgctctcctg
     6661 ttcccgtcct gcggcgtccg tgttgtggtg gaggctttac ccaaatcacc acgtcccgtt
     6721 ccgtgtagac agttcgctcc aagctgggct gtgtgcaaga accccccgtt cagcccgact
     6781 gctgcgcctt atccggtaac tatcatcttg agtccaaccc ggaaagacac gacaaaacgc
     6841 cactggcagc agccattggt aactgagaat tagtggattt agatatcgag agtcttgaag
     6901 tggtggccta acagaggcta cactgaaagg acagtatttg gtatctgcgc tccactaaag
     6961 ccagttacca ggttaagcag ttccccaact gacttaacct tcgatcaaac cgcctcccca
     7021 ggcggttttt tcgtttacag agcaggagat tacgacgatc gtaaaaggat ctcaagaaga
     7081 tcctttacgg attcccgaca ccatcactct agatttcagt gcaatttatc tcttcaaatg
     7141 tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag tcatgccccg
     7201 cgcccaccgg aaggagctga ctgggttgct cctagggtct gattcgttac caattatgac
     7261 aacttgacgg ctacatcatt cactttttct tcaca
//
