LOCUS       sequence-217849-        4193 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 4193)
  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 4193)
  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..4193
                     /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"
     primer_bind     complement(959..977)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      973..1020
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1250..2065)
                     /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(1250..2065)
                     /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     1973..1992
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2066..2157)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2175..2810)
                     /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(2320..2339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3008..4090)
                     /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     4052..4071
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4091..4168)
                     /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 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1021 ctgaaacctc aggcatttga gaagcacacg gtcacactgc ttccggtagt caataaaccg
     1081 gtaaaccagc aatagacata agcggctatt taacgaccct gccctgaacc gacgacaagc
     1141 tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1201 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     1261 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     1321 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     1381 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     1441 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     1501 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     1561 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     1621 gacgaaatac gcggtcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     1681 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     1741 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     1801 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     1861 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     1921 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     1981 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     2041 acgtttcccg ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc
     2101 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2161 gcatgctagc gcagaaacgt cctagaagat gccaggagga tacttagcag agagacaata
     2221 aggccggagc gaagccgttt ttccataggc tccgcccccc tgacgaacat cacgaaatct
     2281 gacgctcaaa tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2341 ctgatggctc cctcttgcgc tctcctgttc ccgtcctgcg gcgtccgtgt tgtggtggag
     2401 gctttaccca aatcaccacg tcccgttccg tgtagacagt tcgctccaag ctgggctgtg
     2461 tgcaagaacc ccccgttcag cccgactgct gcgccttatc cggtaactat catcttgagt
     2521 ccaacccgga aagacacgac aaaacgccac tggcagcagc cattggtaac tgagaattag
     2581 tggatttaga tatcgagagt cttgaagtgg tggcctaaca gaggctacac tgaaaggaca
     2641 gtatttggta tctgcgctcc actaaagcca gttaccaggt taagcagttc cccaactgac
     2701 ttaaccttcg atcaaaccgc ctccccaggc ggttttttcg tttacagagc aggagattac
     2761 gacgatcgta aaaggatctc aagaagatcc tttacggatt cccgacacca tcactctaga
     2821 tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt
     2881 tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     2941 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     3001 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3061 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     3121 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     3181 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     3241 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
     3301 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     3361 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     3421 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     3481 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     3541 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     3601 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     3661 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     3721 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     3781 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     3841 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     3901 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     3961 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     4021 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     4081 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     4141 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atg
//
