LOCUS       sequence-60751-e        5958 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 5958)
  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 5958)
  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-G1Tt
FEATURES             Location/Qualifiers
     source          1..5958
                     /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    2439..2455
                     /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)."
     promoter        2586..3027
                     /label=TPGI promoter
                     /note="S. cerevisiae GAL1 promoter modified to contain two 
                     copies of the tet operator (Ellis et al., 2009)"
     protein_bind    2586..2703
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     protein_bind    2911..2929
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2932..2950
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2998..3021
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3051..3674
                     /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(3124..3143)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3707..3896
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3968..3985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4139..4727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4219..4238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4898..5689,5690..5758))
                     /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     5521..5540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5759..5863)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5931..5949
                     /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 agacccactt
     2101 tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa taagaaggct
     2161 ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg cggcatacta
     2221 tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc ttccaatacg
     2281 caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc actagtgcgg
     2341 ccgcccttta gtgagggttg aattcgaatt ttcaaaaatt cttacttttt ttttggatgg
     2401 acgcaaagaa gtttaataat catattacat gtgtgaaatt gttatccgct cacaattcca
     2461 cacaacatat ctaatcttac ttatatgttg tggaaatgta aagagcccca ttatcttagc
     2521 ctaaaaaaac cttctctttg gaactttcag taatacgctt aactgctcat tgctatattg
     2581 aagtacggat tagaagccgc cgagcgggtg acagccctcc gaaggaagac tctcctccgt
     2641 gcgtcctcgt cttcaccggt cgcgttcctg aaacgcagat gtgcctcgcg ccgcactgct
     2701 ccgaacaata aagattctac aatactagct tttatggtta tgaagaggaa aaattggcag
     2761 taacctggcc ccacaaacct tcaaatgaac gaatcaaatt aacaaccata ggatgataat
     2821 gcgattagtt ttttagcctt atttctgggg taattaatca gcgaagcgat gatttttgat
     2881 ctattaacag atatataaat gcaaaaactg tccctatcag tgatagagat ctccctatca
     2941 gtgatagaga tattacttct tattcaaatg taataaaagt atcaacaaaa aattgttaat
     3001 atacctctat actttaacgt caaggagaaa aaaccccgga tcctattaaa atgtctagat
     3061 tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag
     3121 gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc
     3181 atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc
     3241 atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta
     3301 aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac
     3361 ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag
     3421 gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt
     3481 gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg
     3541 atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc
     3601 cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg
     3661 aaagtgggtc ttaactcgag taagcttggt accgcggcta gctaagatcc gctctaaccg
     3721 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt
     3781 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt
     3841 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcc
     3901 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3961 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4021 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4081 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4141 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4201 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4261 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4321 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4381 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4441 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4501 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4561 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4621 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4681 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4741 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4801 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4861 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4921 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4981 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5041 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5101 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5161 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5221 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5281 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5341 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5401 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5461 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5521 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5581 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5641 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5701 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5761 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5821 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5881 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     5941 tcacgaggcc ctttcgtc
//
