LOCUS       sequence-305140-        4415 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cL control for toggle switch experiment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 3489 to 3523)
  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 4415)
  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: pJ2076.2_cL
FEATURES             Location/Qualifiers
     source          1..4415
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    120..139
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    206..225
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             230..252
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             260..973
                     /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"
     terminator      1646..1673
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1861..2676)
                     /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(1861..2676)
                     /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     2584..2603
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2677..2768)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2786..3421)
                     /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(2931..2950)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3489..3523
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     promoter        4235..4269
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    join(4413..4415,1..17)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
ORIGIN
        1 cactgccgta taggcagcat cttagagtat gtagtttcag agctatgctg gaaacagcat
       61 agcaagttga aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgcg
      121 ttcactgccg tataggcagt cgctgggacg cccgggatcc aagagatttc tacacgattg
      181 agcactgtct cagcctgcgg tccgggttca ctgccgtata ggcagtaatt ttgtttaact
      241 ttaagaagga gatatacata tgcgtaaagg cgaagaactg tttaccggtg tggttccgat
      301 tctggtggaa ctggacggcg atgttaatgg tcataaattc agtgttcgcg gcgaaggtga
      361 aggcgatgcg acgaacggca aactgaccct gaaatttatc tgcaccacgg gtaaactgcc
      421 ggtcccgtgg ccgacgctgg tgaccacgct gacctatggc gttcaatgtt ttgcgcgtta
      481 cccggatcac atgaaacagc acgacttttt caaatcggcc atgccggaag gctatgtgca
      541 ggaacgtacg attagcttta aagacgatgg tacgtataaa acccgcgcgg aagtgaaatt
      601 cgaaggcgat accctggtta accgtatcga actgaaaggt atcgatttca aagaagacgg
      661 caatattctg ggtcataaac tggaatataa cttcaattcc cacaacgtgt acatcaccgc
      721 ggataaacag aaaaacggca ttaaagccaa tttcaaaatc cgccataatg tggaagatgg
      781 tagcgttcag ctggccgacc actatcagca aaacacgccg attggtgatg gcccggtcct
      841 gctgccggac aatcactacc tgagtaccca gtccgtgctg tcaaaagatc cgaacgaaaa
      901 acgtgaccac atggtcctgc tggaatttgt gacggctgcg ggtatcaccc acggcatgga
      961 cgaactgtat aaaatgtccc gccgtaatac tgacgccatc acaatccaca gcatcctgga
     1021 ttggattgaa gactaataat cgctgggacg cccgcctgca ggctcggtac caaattccag
     1081 aaaagaggcc tcccgaaagg ggggcctttt ttcgttttgg tcctaataga taaaggatag
     1141 gtctggtagt gttgttcgtt ctcgcaggta aatcaataat actcagcagt tccgtagact
     1201 tttcagtggg acagggtagc gataacagat agattgtaat aagacacagt aggtgctcgt
     1261 agttgcgtga agagaaccgc tcaggaaatc cagtcagaag tattggtaat cgttgaaaac
     1321 tcagtcgacg cacttactga agacgtccta ttacactcgt cgttggaaac tgaagatgcg
     1381 gccgcggaaa cacagaaaaa agcccgcacc tgacagtgcg ggcttttttt ttcgaccaaa
     1441 ggtagcgaac gacgagtcac tgttgaggat aaatactttc tctactaggc gcctgttaca
     1501 caggtcctca gcggcgcgcc tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     1561 ggagcggatt tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     1621 taaactgcca ggcatcaaac taagcagaag gccatcctga cggatggcct ttttgcgttt
     1681 cagatctacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac
     1741 cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga
     1801 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1861 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1921 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1981 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2041 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2101 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2161 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2221 cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga
     2281 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2341 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2401 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2461 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2521 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2581 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2641 cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg
     2701 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2761 taaacaaata ggcatgctag cgcagaaacg tcctagaaga tgccaggagg atacttagca
     2821 gagagacaat aaggccggag cgaagccgtt tttccatagg ctccgccccc ctgacgaaca
     2881 tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca
     2941 ggcgtttccc cctgatggct ccctcttgcg ctctcctgtt cccgtcctgc ggcgtccgtg
     3001 ttgtggtgga ggctttaccc aaatcaccac gtcccgttcc gtgtagacag ttcgctccaa
     3061 gctgggctgt gtgcaagaac cccccgttca gcccgactgc tgcgccttat ccggtaacta
     3121 tcatcttgag tccaacccgg aaagacacga caaaacgcca ctggcagcag ccattggtaa
     3181 ctgagaatta gtggatttag atatcgagag tcttgaagtg gtggcctaac agaggctaca
     3241 ctgaaaggac agtatttggt atctgcgctc cactaaagcc agttaccagg ttaagcagtt
     3301 ccccaactga cttaaccttc gatcaaaccg cctccccagg cggttttttc gtttacagag
     3361 caggagatta cgacgatcgt aaaaggatct caagaagatc ctttacggat tcccgacacc
     3421 atcactctag agaattcata ggatagattc tggaaacttt accgtccgag ctccagcctg
     3481 cggtccggtt gacggctagc tcagtcctag gtacagtgct agctcgctgg gacgcccgag
     3541 agccgaatcg cacttattta cagtagttca gcctgcggtc cggccataca tactctaaga
     3601 tgtgtctcgc tgggacgccc ggggactaca cttacgaaac tattgattgc tcagcctgcg
     3661 gtccggtgta agagaaatcc acgaaggaaa aggtgaaagc agaacagata atcagtaata
     3721 gtagcaactc aaccacagga cacttccctc actccgaacg aatcacaata cgctccaatc
     3781 ttcaactaca cgataaaact ggtaatccca acaacctgac tcacactcta ctttctcaac
     3841 gaggaacttc ttattctgat acaatctcgc tgggacgccc gctcgagcaa taaacagttg
     3901 atagggcttc tccgttacca tggttcagcc aaaaaactta agaccgccgg tcttgtccac
     3961 taccttgcag taatgcggtg gacaggatcg gcggttttct tttctcttct caattcttct
     4021 gacctgtaac gaataataga tagtaaagta gtctccgatt gagttttctc tgccgagtcc
     4081 cacccagttc tgtgatttca gtaagttggt aattgataca ctgttgcgag aactgctgcc
     4141 tggtagtaga taggttgtta ttgagtaaga aggtaaagtg aacgaaatcc ctgaaactga
     4201 gactgtagaa aataagcttc agcctgcggt ccggttgaca gctagctcag tcctaggtac
     4261 tgtgctagct cgctgggacg cccgagatag ccgttacaca ggtgacactt atttcagcct
     4321 gcggtccggc cacactagag tctagcttga gatcgctggg acgcccgtga acagttgctc
     4381 tgattgaaac cacgattcag cctgcggtcc gggtt
//
