LOCUS       sequence-261786-        4298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of 3WJ repressor index 19 switch RNA with a 
            GFP-ASV reporter via the Ptet* promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4298)
  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 4298)
  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_Ptet*_3WJrep_N19_switch
FEATURES             Location/Qualifiers
     source          1..4298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        3..52
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             170..883
                     /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             170..883
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(200..228)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     823..843
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             complement(1248..2063)
                     /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(1248..2063)
                     /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     1971..1990
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2064..2155)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2173..2808)
                     /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(2318..2337)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3006..4088)
                     /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     4050..4069
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4089..4166)
                     /gene="lacI"
                     /label=lacI promoter
     protein_bind    4245..4263
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4270..4288
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca
       61 aataattttg tttaaactaa tcagatctac ttgttatagt tatgaacaga ggagacataa
      121 catgaacaag cacctaacaa gactaatcaa cctggcggca gcgcaaaaga tgcgtaaagg
      181 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
      241 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
      301 taaatttatt tgcactactg gaaaactacc tgttccgtgg ccaacacttg tcactacttt
      361 cggttatggt gttcaatgct ttgcgagata cccagatcac atgaaacagc atgacttttt
      421 caagagtgcc atgcccgaag gttacgtaca ggaaagaact atatttttca aagatgacgg
      481 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
      541 gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa
      601 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa
      661 cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca
      721 aaatactccg attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
      781 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
      841 aaccgctgct gggattacac atggcatgga tgaactatac aaaaggcctg cagcaaacga
      901 cgaaaactac gctgcatcag tttaataaac ttaagaccgc cggtcttgtc cactaccttg
      961 cagtaatgcg gtggacagga tcggcggttt tcttttctcg gcaccggcac gttaatacgc
     1021 aaacactgtg gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
     1081 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgacaagctg
     1141 acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     1201 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     1261 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     1321 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     1381 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     1441 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     1501 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     1561 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     1621 cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     1681 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     1741 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     1801 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     1861 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     1921 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     1981 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     2041 gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag catttatcag
     2101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggc
     2161 atgctagcgc agaaacgtcc tagaagatgc caggaggata cttagcagag agacaataag
     2221 gccggagcga agccgttttt ccataggctc cgcccccctg acgaacatca cgaaatctga
     2281 cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2341 gatggctccc tcttgcgctc tcctgttccc gtcctgcggc gtccgtgttg tggtggaggc
     2401 tttacccaaa tcaccacgtc ccgttccgtg tagacagttc gctccaagct gggctgtgtg
     2461 caagaacccc ccgttcagcc cgactgctgc gccttatccg gtaactatca tcttgagtcc
     2521 aacccggaaa gacacgacaa aacgccactg gcagcagcca ttggtaactg agaattagtg
     2581 gatttagata tcgagagtct tgaagtggtg gcctaacaga ggctacactg aaaggacagt
     2641 atttggtatc tgcgctccac taaagccagt taccaggtta agcagttccc caactgactt
     2701 aaccttcgat caaaccgcct ccccaggcgg ttttttcgtt tacagagcag gagattacga
     2761 cgatcgtaaa aggatctcaa gaagatcctt tacggattcc cgacaccatc actctagatt
     2821 tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg atataagttg
     2881 taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     2941 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     3001 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3061 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     3121 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     3181 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     3241 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg
     3301 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     3361 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     3421 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     3481 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     3541 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     3601 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     3661 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     3721 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     3781 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     3841 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     3901 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     3961 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     4021 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     4081 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     4141 aaggttttgc gccattcgat ggtgtccggg atctccgacg ctctccctta tgggccaact
     4201 aacccacgtg tgattgtagc tattttcagc aggacgcact gacctcccta tcagtgatag
     4261 agattgacat ccctatcagt gatagagata ctgagcac
//
