LOCUS       sequence-211516-        4192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Toehold switch sensor to detect the tcdB mRNA from toxigenic C. 
            difficile with GFP output.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4192)
  AUTHORS   Takahashi MK, Tan X, Dy AJ, Braff D, Akana RT, Furuta Y, Donghia N, 
            Ananthakrishnan A, Collins JJ
  TITLE     A low-cost paper-based synthetic biology platform for analyzing gut 
            microbiota and host biomarkers.
  JOURNAL   Nat Commun. 2018 Aug 21;9(1):3347. doi: 10.1038/s41467-018-05864-4.
  PUBMED    30131493
REFERENCE   2  (bases 1 to 4192)
  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: "2018-08-21"; volume: "9(1):3347. doi"; pages: " 
            10.1038/s41467-018-05864-4"
COMMENT     Sequence Label: C. difficile toxin B mRNA toehold switch sensor
FEATURES             Location/Qualifiers
     source          1..4192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             151..864
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             151..864
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(181..209)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     804..824
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      972..1019
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1249..2064)
                     /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(1249..2064)
                     /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     1972..1991
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2065..2156)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2174..2809)
                     /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(2319..2338)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3007..4089)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4051..4070
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4090..4167)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagtctaacg ctgctctggg ctaactgtcg cgctaatacg actcactata gggtgttttt
       61 tcagtttcta caaatataga ttctccagtg gactttagaa cagaggagat aaagatgact
      121 ggagaatcta acctggcggc agcgcaaaag atgcgtaaag gagaagaact tttcactgga
      181 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
      241 ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact
      301 ggaaaactac ctgttccgtg gccaacactt gtcactactt tcggttatgg tgttcaatgc
      361 tttgcgagat acccagatca catgaaacag catgactttt tcaagagtgc catgcccgaa
      421 ggttacgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct
      481 gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt
      541 aaagaagatg gaaacattct tggacacaaa ttggaataca actataactc acacaatgta
      601 tacatcatgg cagacaaaca aaagaatgga atcaaagtta acttcaaaat tagacacaac
      661 attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc gattggcgat
      721 ggccctgtcc ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat
      781 cccaacgaaa agagagacca catggtcctt cttgagtttg taaccgctgc tgggattaca
      841 catggcatgg atgaactata caaaaggcct gcagcaaacg acgaaaacta cgctgcatca
      901 gtttaataag ataaaccaga gcggcacggc aagcagagta tacgagattc ggtagccacc
      961 gctgagcata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1021 tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg
     1081 taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgacaagct
     1141 gacgaccggg tctccgcaag tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1201 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     1261 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     1321 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1381 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1441 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1501 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     1561 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     1621 acgaaatacg cggtcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     1681 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     1741 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     1801 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     1861 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     1921 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     1981 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     2041 cgtttcccgt tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca
     2101 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2161 catgctagcg cagaaacgtc ctagaagatg ccaggaggat acttagcaga gagacaataa
     2221 ggccggagcg aagccgtttt tccataggct ccgcccccct gacgaacatc acgaaatctg
     2281 acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2341 tgatggctcc ctcttgcgct ctcctgttcc cgtcctgcgg cgtccgtgtt gtggtggagg
     2401 ctttacccaa atcaccacgt cccgttccgt gtagacagtt cgctccaagc tgggctgtgt
     2461 gcaagaaccc cccgttcagc ccgactgctg cgccttatcc ggtaactatc atcttgagtc
     2521 caacccggaa agacacgaca aaacgccact ggcagcagcc attggtaact gagaattagt
     2581 ggatttagat atcgagagtc ttgaagtggt ggcctaacag aggctacact gaaaggacag
     2641 tatttggtat ctgcgctcca ctaaagccag ttaccaggtt aagcagttcc ccaactgact
     2701 taaccttcga tcaaaccgcc tccccaggcg gttttttcgt ttacagagca ggagattacg
     2761 acgatcgtaa aaggatctca agaagatcct ttacggattc ccgacaccat cactctagat
     2821 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     2881 gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     2941 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     3001 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3061 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     3121 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     3181 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     3241 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac
     3301 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     3361 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3421 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     3481 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     3541 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     3601 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     3661 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     3721 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     3781 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     3841 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     3901 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     3961 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     4021 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     4081 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     4141 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tg
//
