LOCUS       sequence-261831-        3411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of second-generation toehold repressor 
            index 2 trigger RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3411)
  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 3411)
  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_trigger
FEATURES             Location/Qualifiers
     source          1..3411
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      81..127
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(298..1089)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1090..1181)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1229..1967)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(2177..3259)
                     /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     3221..3240
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3260..3337)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(3395..3411,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3395..3411,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgatagc tatacctgga tagctatcgc attcttctta ttctcgtctc tttccacttt
       61 attcttactt tcactttact tagcataacc ccttggggcc tctaaacggg tcttgagggg
      121 ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
      181 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
      241 gaccgggtca tcgtggccgg atcttgcggc ccctcggctt gaacgaattg ttagacatta
      301 tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc
      361 tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat
      421 gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg
      481 cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg
      541 ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc
      601 aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc
      661 aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg
      721 ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt
      781 agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg
      841 gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg
      901 ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg
      961 cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag
     1021 atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc
     1081 ttccctcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1141 gagcggatac atatttgaat gtatttagaa aaataaacaa atagctagct cactcggtcg
     1201 ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt tacccacaga
     1261 ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc gccggtggcg
     1321 tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt ttccggtgca
     1381 tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac ccgacaggac
     1441 ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg ttccgaccct
     1501 gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc gctttctcat
     1561 agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg ggctgtaagc
     1621 aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca cttgagtcca
     1681 acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg gagttcgcag
     1741 aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt ccggctacac
     1801 tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt taagcagttc
     1861 cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg tttacagggc
     1921 aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctactgaac
     1981 cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     2041 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     2101 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     2161 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2221 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     2281 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2341 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2401 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     2461 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     2521 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     2581 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     2641 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     2701 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     2761 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     2821 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     2881 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     2941 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     3001 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     3061 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3121 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3181 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3241 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3301 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     3361 tgaacgtgta cgggctatct ggctttcgtt gcgctaatac gactcactat a
//
