LOCUS       sequence-263373-        4231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of second-generation toehold repressor 
            index 2 switch RNA with a GFP-ASV reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4231)
  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 4231)
  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: pJK_ToeRepG2_N02_switch
FEATURES             Location/Qualifiers
     source          1..4231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             140..853
                     /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             140..853
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(170..198)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     793..813
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(948..966)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      962..1009
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1239..2054)
                     /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(1239..2054)
                     /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     1962..1981
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2055..2146)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2164..2799)
                     /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(2309..2328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2997..4079)
                     /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     4041..4060
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4080..4157)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4216..4231,1..4)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4216..4231,1..3)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agggaaagtg aaagtaagaa taaagtggaa agagacgaga ataagaagag acatcatatc
       61 ttcttattct cgtctctttc tactttataa acagaggaga ataaagatga aagagacgaa
      121 cctggcggca gcgcaaaaga tgcgtaaagg agaagaactt ttcactggag ttgtcccaat
      181 tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga
      241 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
      301 tgttccgtgg ccaacacttg tcactacttt cggttatggt gttcaatgct ttgcgagata
      361 cccagatcac atgaaacagc atgacttttt caagagtgcc atgcccgaag gttacgtaca
      421 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
      481 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
      541 aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc
      601 agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg
      661 aagcgttcaa ctagcagacc attatcaaca aaatactccg attggcgatg gccctgtcct
      721 tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa
      781 gagagaccac atggtccttc ttgagtttgt aaccgctgct gggattacac atggcatgga
      841 tgaactatac aaaaggcctg cagcaaacga cgaaaactac gctgcatcag tttaataaga
      901 taaaccagag cggcacggca agcagagtat acgagattcg gtagccaccg ctgagcaata
      961 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaacctca
     1021 ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca
     1081 atagacataa gcggctattt aacgaccctg ccctgaaccg acgacaagct gacgaccggg
     1141 tctccgcaag tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1201 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     1261 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     1321 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     1381 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     1441 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     1501 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     1561 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     1621 cggtcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     1681 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     1741 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     1801 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1861 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1921 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1981 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     2041 tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2101 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg catgctagcg
     2161 cagaaacgtc ctagaagatg ccaggaggat acttagcaga gagacaataa ggccggagcg
     2221 aagccgtttt tccataggct ccgcccccct gacgaacatc acgaaatctg acgctcaaat
     2281 cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc tgatggctcc
     2341 ctcttgcgct ctcctgttcc cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa
     2401 atcaccacgt cccgttccgt gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc
     2461 cccgttcagc ccgactgctg cgccttatcc ggtaactatc atcttgagtc caacccggaa
     2521 agacacgaca aaacgccact ggcagcagcc attggtaact gagaattagt ggatttagat
     2581 atcgagagtc ttgaagtggt ggcctaacag aggctacact gaaaggacag tatttggtat
     2641 ctgcgctcca ctaaagccag ttaccaggtt aagcagttcc ccaactgact taaccttcga
     2701 tcaaaccgcc tccccaggcg gttttttcgt ttacagagca ggagattacg acgatcgtaa
     2761 aaggatctca agaagatcct ttacggattc ccgacaccat cactctagat ttcagtgcaa
     2821 tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca
     2881 tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     2941 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     3001 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3061 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     3121 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     3181 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     3241 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     3301 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     3361 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     3421 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     3481 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     3541 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     3601 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     3661 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     3721 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     3781 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     3841 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     3901 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     3961 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     4021 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     4081 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     4141 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgaagtctaa cgctgctctg
     4201 ggctaactgt cgcgctaata cgactcacta t
//
