LOCUS       sequence-305139-        4361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Toggle switch (TS).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 4271 to 4305)
  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 4361)
  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_TS
FEATURES             Location/Qualifiers
     source          1..4361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    88..107
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    214..233
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        602..636
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
     protein_bind    780..799
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    902..921
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    988..1007
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             1012..1034
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1042..1755
                     /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      2428..2455
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2643..3458)
                     /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(2643..3458)
                     /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     3366..3385
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(3459..3550)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3568..4203)
                     /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(3713..3732)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4271..4305
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
ORIGIN
        1 ccggccatac atactctaag atgtgtctcg ctgggacgcc cggggactac acttacgaaa
       61 ctattgattg ctcagcctgc ggtccgggtt cactgccgta taggcagtct caagctagac
      121 tctagtggtt tcagagctat gctggaaaca gcatagcaag ttgaaataag gctagtccgt
      181 tatcaacttg aaaaagtggc accgagtcgg tgcgttcact gccgtatagg cagtcgctgg
      241 gacgcccgct cgagcaataa acagttgata gggcttctcc gttaccatgg ttcagccaaa
      301 aaacttaaga ccgccggtct tgtccactac cttgcagtaa tgcggtggac aggatcggcg
      361 gttttctttt ctcttctcaa ttcttctgac ctgtaacgaa taatagatag taaagtagtc
      421 tccgattgag ttttctctgc cgagtcccac ccagttctgt gatttcagta agttggtaat
      481 tgatacactg ttgcgagaac tgctgcctgg tagtagatag gttgttattg agtaagaagg
      541 taaagtgaac gaaatccctg aaactgagac tgtagaaaat aagcttcagc ctgcggtccg
      601 gttgacagct agctcagtcc taggtactgt gctagctcgc tgggacgccc gagatagccg
      661 ttacacaggt gacacttatt tcagcctgcg gtccggccac actagagtct agcttgagat
      721 cgctgggacg cccgtgaaca gttgctctga ttgaaaccac gattcagcct gcggtccggg
      781 ttcactgccg tataggcagc atcttagagt atgtagtttc agagctatgc tggaaacagc
      841 atagcaagtt gaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      901 cgttcactgc cgtataggca gtcgctggga cgcccgggat ccaagagatt tctacacgat
      961 tgagcactgt ctcagcctgc ggtccgggtt cactgccgta taggcagtaa ttttgtttaa
     1021 ctttaagaag gagatataca tatgcgtaaa ggcgaagaac tgtttaccgg tgtggttccg
     1081 attctggtgg aactggacgg cgatgttaat ggtcataaat tcagtgttcg cggcgaaggt
     1141 gaaggcgatg cgacgaacgg caaactgacc ctgaaattta tctgcaccac gggtaaactg
     1201 ccggtcccgt ggccgacgct ggtgaccacg ctgacctatg gcgttcaatg ttttgcgcgt
     1261 tacccggatc acatgaaaca gcacgacttt ttcaaatcgg ccatgccgga aggctatgtg
     1321 caggaacgta cgattagctt taaagacgat ggtacgtata aaacccgcgc ggaagtgaaa
     1381 ttcgaaggcg ataccctggt taaccgtatc gaactgaaag gtatcgattt caaagaagac
     1441 ggcaatattc tgggtcataa actggaatat aacttcaatt cccacaacgt gtacatcacc
     1501 gcggataaac agaaaaacgg cattaaagcc aatttcaaaa tccgccataa tgtggaagat
     1561 ggtagcgttc agctggccga ccactatcag caaaacacgc cgattggtga tggcccggtc
     1621 ctgctgccgg acaatcacta cctgagtacc cagtccgtgc tgtcaaaaga tccgaacgaa
     1681 aaacgtgacc acatggtcct gctggaattt gtgacggctg cgggtatcac ccacggcatg
     1741 gacgaactgt ataaaatgtc ccgccgtaat actgacgcca tcacaatcca cagcatcctg
     1801 gattggattg aagactaata atcgctggga cgcccgcctg caggctcggt accaaattcc
     1861 agaaaagagg cctcccgaaa ggggggcctt ttttcgtttt ggtcctaata gataaaggat
     1921 aggtctggta gtgttgttcg ttctcgcagg taaatcaata atactcagca gttccgtaga
     1981 cttttcagtg ggacagggta gcgataacag atagattgta ataagacaca gtaggtgctc
     2041 gtagttgcgt gaagagaacc gctcaggaaa tccagtcaga agtattggta atcgttgaaa
     2101 actcagtcga cgcacttact gaagacgtcc tattacactc gtcgttggaa actgaagatg
     2161 cggccgcgga aacacagaaa aaagcccgca cctgacagtg cgggcttttt ttttcgacca
     2221 aaggtagcga acgacgagtc actgttgagg ataaatactt tctctactag gcgcctgtta
     2281 cacaggtcct cagcggcgcg cctttgtcgg tgaacgctct cctgagtagg acaaatccgc
     2341 cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     2401 cataaactgc caggcatcaa actaagcaga aggccatcct gacggatggc ctttttgcgt
     2461 ttcagatcta ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga
     2521 accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga aatgtgcgcg
     2581 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     2641 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     2701 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     2761 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     2821 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     2881 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2941 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     3001 tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca aacaggaatc
     3061 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     3121 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     3181 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     3241 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     3301 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     3361 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     3421 ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat
     3481 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3541 aataaacaaa taggcatgct agcgcagaaa cgtcctagaa gatgccagga ggatacttag
     3601 cagagagaca ataaggccgg agcgaagccg tttttccata ggctccgccc ccctgacgaa
     3661 catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac
     3721 caggcgtttc cccctgatgg ctccctcttg cgctctcctg ttcccgtcct gcggcgtccg
     3781 tgttgtggtg gaggctttac ccaaatcacc acgtcccgtt ccgtgtagac agttcgctcc
     3841 aagctgggct gtgtgcaaga accccccgtt cagcccgact gctgcgcctt atccggtaac
     3901 tatcatcttg agtccaaccc ggaaagacac gacaaaacgc cactggcagc agccattggt
     3961 aactgagaat tagtggattt agatatcgag agtcttgaag tggtggccta acagaggcta
     4021 cactgaaagg acagtatttg gtatctgcgc tccactaaag ccagttacca ggttaagcag
     4081 ttccccaact gacttaacct tcgatcaaac cgcctcccca ggcggttttt tcgtttacag
     4141 agcaggagat tacgacgatc gtaaaaggat ctcaagaaga tcctttacgg attcccgaca
     4201 ccatcactct agagaattca taggatagat tctggaaact ttaccgtccg agctccagcc
     4261 tgcggtccgg ttgacggcta gctcagtcct aggtacagtg ctagctcgct gggacgcccg
     4321 agagccgaat cgcacttatt tacagtagtt cagcctgcgg t
//
