LOCUS       sequence-261728-        4191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of 3WJ repressor index 10 switch RNA with 
            a GFP-ASV reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4191)
  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 4191)
  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: pYZ_3WJrep_N10_switch
FEATURES             Location/Qualifiers
     source          1..4191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             99..812
                     /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             99..812
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(129..157)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     752..772
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(907..925)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      921..968
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1198..2013)
                     /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(1198..2013)
                     /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     1921..1940
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2014..2105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2123..2758)
                     /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(2268..2287)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2956..4038)
                     /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     4000..4019
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4039..4116)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4176..4191,1..4)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4176..4191,1..3)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agggtagtaa gattagatat tgttatagtt atgaacagag gagacataac atgaacaagt
       61 acaagaaccg aaacgagaac ctggcggcag cgcaaaagat gcgtaaagga gaagaacttt
      121 tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt
      181 ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt
      241 gcactactgg aaaactacct gttccgtggc caacacttgt cactactttc ggttatggtg
      301 ttcaatgctt tgcgagatac ccagatcaca tgaaacagca tgactttttc aagagtgcca
      361 tgcccgaagg ttacgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga
      421 cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag ttaaaaggta
      481 ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac tataactcac
      541 acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta
      601 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccga
      661 ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt
      721 cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta accgctgctg
      781 ggattacaca tggcatggat gaactataca aaaggcctgc agcaaacgac gaaaactacg
      841 ctgcatcagt ttaataagat aaaccagagc ggcacggcaa gcagagtata cgagattcgg
      901 tagccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
      961 gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca
     1021 ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga
     1081 cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc
     1141 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1201 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1261 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1321 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     1381 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1441 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     1501 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     1561 ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     1621 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     1681 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     1741 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     1801 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     1861 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     1921 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     1981 agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag
     2041 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2101 acaaataggc atgctagcgc agaaacgtcc tagaagatgc caggaggata cttagcagag
     2161 agacaataag gccggagcga agccgttttt ccataggctc cgcccccctg acgaacatca
     2221 cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc
     2281 gtttccccct gatggctccc tcttgcgctc tcctgttccc gtcctgcggc gtccgtgttg
     2341 tggtggaggc tttacccaaa tcaccacgtc ccgttccgtg tagacagttc gctccaagct
     2401 gggctgtgtg caagaacccc ccgttcagcc cgactgctgc gccttatccg gtaactatca
     2461 tcttgagtcc aacccggaaa gacacgacaa aacgccactg gcagcagcca ttggtaactg
     2521 agaattagtg gatttagata tcgagagtct tgaagtggtg gcctaacaga ggctacactg
     2581 aaaggacagt atttggtatc tgcgctccac taaagccagt taccaggtta agcagttccc
     2641 caactgactt aaccttcgat caaaccgcct ccccaggcgg ttttttcgtt tacagagcag
     2701 gagattacga cgatcgtaaa aggatctcaa gaagatcctt tacggattcc cgacaccatc
     2761 actctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg
     2821 atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg
     2881 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     2941 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3001 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3061 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3121 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3181 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc
     3241 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     3301 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     3361 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     3421 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     3481 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     3541 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     3601 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     3661 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     3721 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     3781 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     3841 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     3901 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     3961 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4021 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4081 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     4141 ggccgggttt agaaatctga agctctagag agcgctaata cgactcacta t
//
