LOCUS Exported 6483 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pDN-T1TCbt SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6483) AUTHORS Nevozhay D, Adams RM, Murphy KF, Josic K, Balazsi G TITLE Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression. JOURNAL Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5123-8. doi: 10.1073/pnas.0809901106. Epub 2009 Mar 11. PUBMED 19279212 REFERENCE 2 (bases 1 to 6483) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6483 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(29..51) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 151..170 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 191..472 /gene="S. cerevisiae TRP1" /label=TRP1 promoter CDS 473..1147 /codon_start=1 /gene="S. cerevisiae TRP1" /product="phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis" /label=TRP1 /note="yeast auxotrophic marker" /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA KK" rep_origin complement(1246..1701) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1383..1404) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 1595..1614 /label=F1ori-R /note="F1 origin, reverse primer" terminator 1779..1944 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind 1865..1889 /label=pHybLex-R /note="ADH terminator, reverse primer" protein_bind 2199..2215 /label=lac operator /bound_moiety="lac repressor encoded by 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)." protein_bind 2346..2463 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" protein_bind 2671..2689 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2692..2710 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2719..2737 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 2758..2781 /label=GAL1 /note="S. cerevisiae GAL1 promoter, forward primer" CDS 2811..3431 /codon_start=1 /gene="tetR from transposon Tn10" /product="tetracycline repressor TetR" /label=TetR /note="TetR binds to the tetracycline operator tetO to inhibit transcription. This inhibition can be relieved by adding tetracycline or doxycycline." /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS" primer_bind complement(2884..2903) /label=Tet-R /note="Tetracycline resistance gene, reverse primer" terminator 4232..4421 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(4493..4510) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4664..5252) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4744..4763) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5423..6283) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 6046..6065 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6284..6388) /gene="bla" /label=AmpR promoter primer_bind 6456..6474 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatagatc aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc 241 gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta 301 ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg 361 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg 421 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt 481 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga 541 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa 601 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata 661 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga 721 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga 781 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc 841 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt 901 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg 961 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc 1021 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg 1081 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa 1141 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gcctatgcgg 1201 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt gtaaacgtta 1261 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg 1321 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg 1381 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa 1441 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg 1501 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt 1561 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg 1621 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta 1681 atgcgccgct acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg 1741 atcggtgcgg gcctcttcgc tattacgcca ctgaattgga gcgacctcat gctatacctg 1801 agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc gttttaaaac 1861 ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt taataataaa 1921 aatcataaat cataagaaat tcgcttattt agaagtgtca acaacgtatc taccaacgat 1981 ttgacccttt tccatctttt cgtaaatttc tggcaaggta gacaagccga caaccttgat 2041 tggagacttg accaaacctc tggcgaagaa ttgttaatta agagctctta actagtgcgg 2101 ccgcccttta gtgagggttg aattcgaatt ttcaaaaatt cttacttttt ttttggatgg 2161 acgcaaagaa gtttaataat catattacat gtgtgaaatt gttatccgct cacaattcca 2221 cacaacatat ctaatcttac ttatatgttg tggaaatgta aagagcccca ttatcttagc 2281 ctaaaaaaac cttctctttg gaactttcag taatacgctt aactgctcat tgctatattg 2341 aagtacggat tagaagccgc cgagcgggtg acagccctcc gaaggaagac tctcctccgt 2401 gcgtcctcgt cttcaccggt cgcgttcctg aaacgcagat gtgcctcgcg ccgcactgct 2461 ccgaacaata aagattctac aatactagct tttatggtta tgaagaggaa aaattggcag 2521 taacctggcc ccacaaacct tcaaatgaac gaatcaaatt aacaaccata ggatgataat 2581 gcgattagtt ttttagcctt atttctgggg taattaatca gcgaagcgat gatttttgat 2641 ctattaacag atatataaat gcaaaaactg tccctatcag tgatagagat ctccctatca 2701 gtgatagaga tattactttc cctatcagtg atagagaagt atcaacaaaa aattgttaat 2761 atacctctat actttaacgt caaggagaaa aaaccccgga tcctattaaa atgtctagat 2821 tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag 2881 gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc 2941 atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc 3001 atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta 3061 aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac 3121 ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag 3181 gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt 3241 gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg 3301 atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc 3361 cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg 3421 aaagtgggtc tggtgacggt gctggtttaa ttaacatggc aactagcggc atggttagta 3481 aaggagaaga aaataacatg gcaatcatta aggagttcat gagattcaaa gttcacatgg 3541 aaggttctgt aaatggacat gaatttgaaa tagaaggtga aggagaagga aggccttatg 3601 aaggaaccca aaccgcgaag ctaaaagtta ctaagggtgg cccattacca tttgcatggg 3661 atatccttag ccctcaattc atgtatgggt caaaggctta tgtcaagcac cccgccgaca 3721 ttccagacta tctaaagtta tcttttcccg aagggtttaa gtgggagcgt gtgatgaact 3781 tcgaagacgg tggcgtggta acagtgactc aggattcgtc cctgcaagat ggtgaattta 3841 tctacaaagt caaattaaga ggaactaact ttccatctga cggcccggtt atgcaaaaaa 3901 agacaatggg ctgggaggcc tcctcagaac gaatgtaccc tgaagatggt gccttgaagg 3961 gtgagattaa acaaagattg aaattgaaag atggtggaca ttatgacgct gaggttaaaa 4021 cgacatacaa agctaagaaa cctgtccagc tcccaggtgc ttacaatgta aatataaaac 4081 ttgatattac atcacataat gaagattata cgatagttga acaatacgaa agggctgagg 4141 ggagacatag tactggtggc atggatgaac tatacaaagg ttctggtacc gcataataac 4201 tcgagtaagc ttggtaccgc ggctagctaa gatccgctct aaccgaaaag gaaggagtta 4261 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt 4321 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta 4381 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa gatccagctg cattaatgaa 4441 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 4501 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4561 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4621 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4681 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4741 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4801 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4861 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4921 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4981 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5041 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5101 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5161 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5221 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5281 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5341 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5401 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 5461 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 5521 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 5581 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 5641 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 5701 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 5761 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 5821 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 5881 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 5941 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 6001 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6061 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6121 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6181 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6241 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6301 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6361 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 6421 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 6481 gtc //