LOCUS       sequence-60863-e        6483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.0809901106"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2009-03-31- 31"; volume: "106"; issue: "13"; pages: "5123-8"
COMMENT     Sequence Label: pDN-T1TCbt
FEATURES             Location/Qualifiers
     source          1..6483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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      complement(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(join(5423..6214,6215..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
//
