LOCUS       sequence-261731-        5569 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7 RNAP-driven expression of 3WJ repressor index 20 trigger RNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5569)
  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 5569)
  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_trigger
FEATURES             Location/Qualifiers
     source          1..5569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      46..92
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(267..272,273..289,290..295))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(322..340)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        408..512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(513..581,582..1373)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(731..750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1544..2132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2033..2052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2286..2303
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2318..2457
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2383..2402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2543..2565
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2559..2750)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4062..5144)
                     /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     5106..5125
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5145..5222)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5428..5447
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(5553..5569,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(5553..5569,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggacgaatt gatttgtcaa ttcgtgcgtg tatatcattc atcattagca taaccccttg
       61 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatatc
      121 ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat ccagggtgac
      181 ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct gactgcgtta
      241 gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct gtcaaacatg
      301 agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
      361 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      421 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      481 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      541 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      601 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      661 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      721 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
      781 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      841 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      901 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      961 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     1021 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
     1081 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     1141 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     1201 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     1261 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1321 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1381 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1441 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1501 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1561 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1621 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1681 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1741 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1801 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1861 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1921 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     1981 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2041 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2101 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2161 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2221 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2281 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     2341 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     2401 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     2461 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     2521 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     2581 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     2641 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     2701 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     2761 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     2821 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     2881 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     2941 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3001 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3061 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3121 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     3181 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     3241 atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg
     3301 gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat
     3361 ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc
     3421 gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt
     3481 tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca atgatcgaag
     3541 ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct
     3601 gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca
     3661 taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt
     3721 cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     3781 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     3841 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     3901 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     3961 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     4021 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     4081 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4141 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     4201 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     4261 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     4321 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     4381 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     4441 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     4501 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     4561 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     4621 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     4681 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     4741 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     4801 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     4861 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     4921 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     4981 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     5041 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     5101 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     5161 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     5221 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     5281 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     5341 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     5401 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     5461 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcggc
     5521 cgggtttaga aatctgaagc tctagagagc gctaatacga ctcactata
//
