LOCUS       sequence-305144-        4419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cR control for toggle switch experiment.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 3631 to 3665)
  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 4419)
  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_cR
FEATURES             Location/Qualifiers
     source          1..4419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    348..367
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             372..394
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             402..1115
                     /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      1788..1815
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(2003..2818)
                     /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(2003..2818)
                     /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     2726..2745
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2819..2910)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2928..3563)
                     /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(3073..3092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3631..3665
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     protein_bind    3809..3828
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    3935..3954
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        4323..4357
                     /label=BBa_J23102
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.86"
ORIGIN
        1 acactagagt ctagcttgag atcgctggga cgcccgtgaa cagttgctct gattgaaacc
       61 acgattcagc ctgcggtccg gtgtaagaga aatccacgaa ggaaaaggtg aaagcagaac
      121 agataatcag taatagtagc aactcaacca caggacactt ccctcactcc gaacgaatca
      181 caatacgctc caatcttcaa ctacacgata aaactggtaa tcccaacaac ctgactcaca
      241 ctctactttc tcaacgagga acttcttatt ctgatacaat ctcgctggga cgcccgggat
      301 ccaagagatt tctacacgat tgagcactgt ctcagcctgc ggtccgggtt cactgccgta
      361 taggcagtaa ttttgtttaa ctttaagaag gagatataca tatgcgtaaa ggcgaagaac
      421 tgtttaccgg tgtggttccg attctggtgg aactggacgg cgatgttaat ggtcataaat
      481 tcagtgttcg cggcgaaggt gaaggcgatg cgacgaacgg caaactgacc ctgaaattta
      541 tctgcaccac gggtaaactg ccggtcccgt ggccgacgct ggtgaccacg ctgacctatg
      601 gcgttcaatg ttttgcgcgt tacccggatc acatgaaaca gcacgacttt ttcaaatcgg
      661 ccatgccgga aggctatgtg caggaacgta cgattagctt taaagacgat ggtacgtata
      721 aaacccgcgc ggaagtgaaa ttcgaaggcg ataccctggt taaccgtatc gaactgaaag
      781 gtatcgattt caaagaagac ggcaatattc tgggtcataa actggaatat aacttcaatt
      841 cccacaacgt gtacatcacc gcggataaac agaaaaacgg cattaaagcc aatttcaaaa
      901 tccgccataa tgtggaagat ggtagcgttc agctggccga ccactatcag caaaacacgc
      961 cgattggtga tggcccggtc ctgctgccgg acaatcacta cctgagtacc cagtccgtgc
     1021 tgtcaaaaga tccgaacgaa aaacgtgacc acatggtcct gctggaattt gtgacggctg
     1081 cgggtatcac ccacggcatg gacgaactgt ataaaatgtc ccgccgtaat actgacgcca
     1141 tcacaatcca cagcatcctg gattggattg aagactaata atcgctggga cgcccgcctg
     1201 caggctcggt accaaattcc agaaaagagg cctcccgaaa ggggggcctt ttttcgtttt
     1261 ggtcctaata gataaaggat aggtctggta gtgttgttcg ttctcgcagg taaatcaata
     1321 atactcagca gttccgtaga cttttcagtg ggacagggta gcgataacag atagattgta
     1381 ataagacaca gtaggtgctc gtagttgcgt gaagagaacc gctcaggaaa tccagtcaga
     1441 agtattggta atcgttgaaa actcagtcga cgcacttact gaagacgtcc tattacactc
     1501 gtcgttggaa actgaagatg cggccgcgga aacacagaaa aaagcccgca cctgacagtg
     1561 cgggcttttt ttttcgacca aaggtagcga acgacgagtc actgttgagg ataaatactt
     1621 tctctactag gcgcctgtta cacaggtcct cagcggcgcg cctttgtcgg tgaacgctct
     1681 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg
     1741 gtggcgggca ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatcct
     1801 gacggatggc ctttttgcgt ttcagatcta ccggtaaacc agcaatagac ataagcggct
     1861 atttaacgac cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac
     1921 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1981 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2041 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2101 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2161 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2221 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2281 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2341 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag
     2401 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2461 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2521 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2581 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2641 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2701 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2761 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac
     2821 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     2881 tatttgaatg tatttagaaa aataaacaaa taggcatgct agcgcagaaa cgtcctagaa
     2941 gatgccagga ggatacttag cagagagaca ataaggccgg agcgaagccg tttttccata
     3001 ggctccgccc ccctgacgaa catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc
     3061 cgacaggact ataaagatac caggcgtttc cccctgatgg ctccctcttg cgctctcctg
     3121 ttcccgtcct gcggcgtccg tgttgtggtg gaggctttac ccaaatcacc acgtcccgtt
     3181 ccgtgtagac agttcgctcc aagctgggct gtgtgcaaga accccccgtt cagcccgact
     3241 gctgcgcctt atccggtaac tatcatcttg agtccaaccc ggaaagacac gacaaaacgc
     3301 cactggcagc agccattggt aactgagaat tagtggattt agatatcgag agtcttgaag
     3361 tggtggccta acagaggcta cactgaaagg acagtatttg gtatctgcgc tccactaaag
     3421 ccagttacca ggttaagcag ttccccaact gacttaacct tcgatcaaac cgcctcccca
     3481 ggcggttttt tcgtttacag agcaggagat tacgacgatc gtaaaaggat ctcaagaaga
     3541 tcctttacgg attcccgaca ccatcactct agagaattca taggatagat tctggaaact
     3601 ttaccgtccg agctccagcc tgcggtccgg ttgacggcta gctcagtcct aggtacagtg
     3661 ctagctcgct gggacgcccg agagccgaat cgcacttatt tacagtagtt cagcctgcgg
     3721 tccggccata catactctaa gatgtgtctc gctgggacgc ccggggacta cacttacgaa
     3781 actattgatt gctcagcctg cggtccgggt tcactgccgt ataggcagtc tcaagctaga
     3841 ctctagtggt ttcagagcta tgctggaaac agcatagcaa gttgaaataa ggctagtccg
     3901 ttatcaactt gaaaaagtgg caccgagtcg gtgcgttcac tgccgtatag gcagtcgctg
     3961 ggacgcccgc tcgagcaata aacagttgat agggcttctc cgttaccatg gttcagccaa
     4021 aaaacttaag accgccggtc ttgtccacta ccttgcagta atgcggtgga caggatcggc
     4081 ggttttcttt tctcttctca attcttctga cctgtaacga ataatagata gtaaagtagt
     4141 ctccgattga gttttctctg ccgagtccca cccagttctg tgatttcagt aagttggtaa
     4201 ttgatacact gttgcgagaa ctgctgcctg gtagtagata ggttgttatt gagtaagaag
     4261 gtaaagtgaa cgaaatccct gaaactgaga ctgtagaaaa taagcttcag cctgcggtcc
     4321 ggttgacagc tagctcagtc ctaggtactg tgctagctcg ctgggacgcc cgagatagcc
     4381 gttacacagg tgacacttat ttcagcctgc ggtccggcc
//
