LOCUS       sequence-261808-        4231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of second-generation toehold repressor 
            index 64 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_N64_switch
FEATURES             Location/Qualifiers
     source          1..4231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             139..852
                     /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             139..852
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(169..197)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     792..812
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(947..965)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      961..1008
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1238..2053)
                     /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(1238..2053)
                     /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     1961..1980
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2054..2145)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2163..2798)
                     /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(2308..2327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2996..4078)
                     /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     4040..4059
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4079..4156)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4215..4231,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4215..4231,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggatgaaag ataagagaat agatgagaga agtgggtttg aatatgatct atctaacatc
       61 ataatcaaac ctacttctcc catctataaa cagaggagaa tagatatgag aagtaggaac
      121 ctggcggcag cgcaaaagat gcgtaaagga gaagaacttt tcactggagt tgtcccaatt
      181 cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa
      241 ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct
      301 gttccgtggc caacacttgt cactactttc ggttatggtg ttcaatgctt tgcgagatac
      361 ccagatcaca tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttacgtacag
      421 gaaagaacta tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt
      481 gaaggtgata cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga
      541 aacattcttg gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca
      601 gacaaacaaa agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga
      661 agcgttcaac tagcagacca ttatcaacaa aatactccga ttggcgatgg ccctgtcctt
      721 ttaccagaca accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag
      781 agagaccaca tggtccttct tgagtttgta accgctgctg ggattacaca tggcatggat
      841 gaactataca aaaggcctgc agcaaacgac gaaaactacg ctgcatcagt ttaataagat
      901 aaaccagagc ggcacggcaa gcagagtata cgagattcgg tagccaccgc tgagcaataa
      961 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag
     1021 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa
     1081 tagacataag cggctattta acgaccctgc cctgaaccga cgacaagctg acgaccgggt
     1141 ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1201 aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca
     1261 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1321 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1381 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1441 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1501 gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1561 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1621 ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1681 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1741 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1801 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1861 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1921 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1981 catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt
     2041 gaatatggct catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     2101 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggc atgctagcgc
     2161 agaaacgtcc tagaagatgc caggaggata cttagcagag agacaataag gccggagcga
     2221 agccgttttt ccataggctc cgcccccctg acgaacatca cgaaatctga cgctcaaatc
     2281 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct gatggctccc
     2341 tcttgcgctc tcctgttccc gtcctgcggc gtccgtgttg tggtggaggc tttacccaaa
     2401 tcaccacgtc ccgttccgtg tagacagttc gctccaagct gggctgtgtg caagaacccc
     2461 ccgttcagcc cgactgctgc gccttatccg gtaactatca tcttgagtcc aacccggaaa
     2521 gacacgacaa aacgccactg gcagcagcca ttggtaactg agaattagtg gatttagata
     2581 tcgagagtct tgaagtggtg gcctaacaga ggctacactg aaaggacagt atttggtatc
     2641 tgcgctccac taaagccagt taccaggtta agcagttccc caactgactt aaccttcgat
     2701 caaaccgcct ccccaggcgg ttttttcgtt tacagagcag gagattacga cgatcgtaaa
     2761 aggatctcaa gaagatcctt tacggattcc cgacaccatc actctagatt tcagtgcaat
     2821 ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg taattctcat
     2881 gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     2941 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     3001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3061 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     3121 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     3181 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     3241 agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg
     3301 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     3361 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     3421 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     3481 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     3541 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     3601 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     3661 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     3721 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     3781 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     3841 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     3901 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     3961 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     4021 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     4081 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     4141 gccattcgat ggtgtccggg atctcgacgc tctcccttat gaagtctaac gctgctctgg
     4201 gctaactgtc gcgctaatac gactcactat a
//
