LOCUS       sequence-263370-        4191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of 3WJ repressor index 20 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_N20_switch
FEATURES             Location/Qualifiers
     source          1..4191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             98..811
                     /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             98..811
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(128..156)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     751..771
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(906..924)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      920..967
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1197..2012)
                     /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(1197..2012)
                     /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     1920..1939
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2013..2104)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2122..2757)
                     /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(2267..2286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2955..4037)
                     /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     3999..4018
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4038..4115)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(4175..4191,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(4175..4191,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggatgaatg atatacactt gttatagtta tgaacagagg agacataaca tgaacaagca
       61 cgaattgact acactaaacc tggcggcagc gcaaaagatg cgtaaaggag aagaactttt
      121 cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc
      181 tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg
      241 cactactgga aaactacctg ttccgtggcc aacacttgtc actactttcg gttatggtgt
      301 tcaatgcttt gcgagatacc cagatcacat gaaacagcat gactttttca agagtgccat
      361 gcccgaaggt tacgtacagg aaagaactat atttttcaaa gatgacggga actacaagac
      421 acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat
      481 tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact ataactcaca
      541 caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagttaact tcaaaattag
      601 acacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa atactccgat
      661 tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat ctgccctttc
      721 gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa ccgctgctgg
      781 gattacacat ggcatggatg aactatacaa aaggcctgca gcaaacgacg aaaactacgc
      841 tgcatcagtt taataagata aaccagagcg gcacggcaag cagagtatac gagattcggt
      901 agccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg
      961 ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa
     1021 taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac
     1081 gacaagctga cgaccgggtc tccgcaagtg gcacttttcg gggaaatgtg cgcggaaccc
     1141 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     1201 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1261 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1321 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1381 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1441 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1501 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1561 tgagcgagac gaaatacgcg gtcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1621 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1681 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1741 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1801 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1861 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1921 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     1981 gagcaagacg tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc
     2041 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     2101 caaataggca tgctagcgca gaaacgtcct agaagatgcc aggaggatac ttagcagaga
     2161 gacaataagg ccggagcgaa gccgtttttc cataggctcc gcccccctga cgaacatcac
     2221 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     2281 tttccccctg atggctccct cttgcgctct cctgttcccg tcctgcggcg tccgtgttgt
     2341 ggtggaggct ttacccaaat caccacgtcc cgttccgtgt agacagttcg ctccaagctg
     2401 ggctgtgtgc aagaaccccc cgttcagccc gactgctgcg ccttatccgg taactatcat
     2461 cttgagtcca acccggaaag acacgacaaa acgccactgg cagcagccat tggtaactga
     2521 gaattagtgg atttagatat cgagagtctt gaagtggtgg cctaacagag gctacactga
     2581 aaggacagta tttggtatct gcgctccact aaagccagtt accaggttaa gcagttcccc
     2641 aactgactta accttcgatc aaaccgcctc cccaggcggt tttttcgttt acagagcagg
     2701 agattacgac gatcgtaaaa ggatctcaag aagatccttt acggattccc gacaccatca
     2761 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     2821 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     2881 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     2941 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3001 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3061 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3121 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     3181 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     3241 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     3301 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3361 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     3421 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     3481 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     3541 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     3601 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     3661 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     3721 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     3781 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     3841 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     3901 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     3961 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4021 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     4081 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     4141 gccgggttta gaaatctgaa gctctagaga gcgctaatac gactcactat a
//
