LOCUS       sequence-263371-        4339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of first-generation toehold repressor index 1 
            switch RNA with a GFP-ASV reporter via the Ptet* promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4339)
  AUTHORS   Kim J, Zhou Y, Carlson PD, Teichmann M, Chaudhary S, Simmel FC, 
            Silver PA, Collins JJ, Lucks JB, Yin P, Green AA
  TITLE     De novo-designed translation-repressing riboregulators for 
            multi-input cellular logic.
  JOURNAL   Nat Chem Biol. 2019 Dec;15(12):1173-1182. doi: 
            10.1038/s41589-019-0388-1. Epub 2019 Nov 4.
  PUBMED    31686032
REFERENCE   2  (bases 1 to 4339)
  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"; doi: 
            "10.1038/s41589-019-0388-1"; journalName: "Nat Chem Biol"; date: 
            "2019-12"; volume: "15"; issue: "12"; pages: "1173-1182"
COMMENT     Sequence Label: pAG_Ptet*_ToeRep_N01_switch
FEATURES             Location/Qualifiers
     source          1..4339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        3..52
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             211..924
                     /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             211..924
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(241..269)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     864..884
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(1289..2104)
                     /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(1289..2104)
                     /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     2012..2031
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2105..2196)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2214..2849)
                     /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(2359..2378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3047..4129)
                     /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     4091..4110
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4130..4207)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    4286..4304
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4311..4329
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca
       61 aataattttg tttaaagaaa tgaagataag atagatgtga tgaagtgcga agttgtaata
      121 acaaataact attactactt cgctcttcat ctcatctata aacagaggag aatagatatg
      181 atgaagagca acctggcggc agcgcaaaag atgcgtaaag gagaagaact tttcactgga
      241 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
      301 ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact
      361 ggaaaactac ctgttccgtg gccaacactt gtcactactt tcggttatgg tgttcaatgc
      421 tttgcgagat acccagatca catgaaacag catgactttt tcaagagtgc catgcccgaa
      481 ggttacgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct
      541 gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt
      601 aaagaagatg gaaacattct tggacacaaa ttggaataca actataactc acacaatgta
      661 tacatcatgg cagacaaaca aaagaatgga atcaaagtta acttcaaaat tagacacaac
      721 attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc gattggcgat
      781 ggccctgtcc ttttaccaga caaccattac ctgtccacac aatctgccct ttcgaaagat
      841 cccaacgaaa agagagacca catggtcctt cttgagtttg taaccgctgc tgggattaca
      901 catggcatgg atgaactata caaaaggcct gcagcaaacg acgaaaacta cgctgcatca
      961 gtttaataaa cttaagaccg ccggtcttgt ccactacctt gcagtaatgc ggtggacagg
     1021 atcggcggtt ttcttttctc ggcaccggca cgttaatacg caaacactgt ggcatttgag
     1081 aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa
     1141 gcggctattt aacgaccctg ccctgaaccg acgacaagct gacgaccggg tctccgcaag
     1201 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1261 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     1321 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1381 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1441 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     1501 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     1561 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     1621 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt
     1681 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     1741 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     1801 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     1861 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     1921 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     1981 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2041 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2101 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     2161 gatacatatt tgaatgtatt tagaaaaata aacaaatagg catgctagcg cagaaacgtc
     2221 ctagaagatg ccaggaggat acttagcaga gagacaataa ggccggagcg aagccgtttt
     2281 tccataggct ccgcccccct gacgaacatc acgaaatctg acgctcaaat cagtggtggc
     2341 gaaacccgac aggactataa agataccagg cgtttccccc tgatggctcc ctcttgcgct
     2401 ctcctgttcc cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt
     2461 cccgttccgt gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc
     2521 ccgactgctg cgccttatcc ggtaactatc atcttgagtc caacccggaa agacacgaca
     2581 aaacgccact ggcagcagcc attggtaact gagaattagt ggatttagat atcgagagtc
     2641 ttgaagtggt ggcctaacag aggctacact gaaaggacag tatttggtat ctgcgctcca
     2701 ctaaagccag ttaccaggtt aagcagttcc ccaactgact taaccttcga tcaaaccgcc
     2761 tccccaggcg gttttttcgt ttacagagca ggagattacg acgatcgtaa aaggatctca
     2821 agaagatcct ttacggattc ccgacaccat cactctagat ttcagtgcaa tttatctctt
     2881 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat
     2941 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     3001 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     3061 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     3121 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     3181 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     3241 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     3301 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     3361 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     3421 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     3481 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3541 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3601 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3661 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     3721 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     3781 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     3841 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     3901 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     3961 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     4021 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     4081 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     4141 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     4201 tggtgtccgg gatctccgac gctctccctt atgggccaac taacccacgt gtgattgtag
     4261 ctattttcag caggacgcac tgacctccct atcagtgata gagattgaca tccctatcag
     4321 tgatagagat actgagcac
//
