LOCUS       sequence-60749-e        6696 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 6696)
  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 6696)
  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-G1TGt
FEATURES             Location/Qualifiers
     source          1..6696
                     /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..3671
                     /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"
     CDS             3696..4412
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      4445..4634
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4706..4723)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4877..5465)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4957..4976)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5636..6427,6428..6496))
                     /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     6259..6278
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6497..6601)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6669..6687
                     /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 tggtgacggt gctggtttaa ttaacatgtc taaaggtgaa gaattattca
     3721 ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac aaattttctg
     3781 tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa tttatttgta
     3841 ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttcggt tatggtgttc
     3901 aatgttttgc gagataccca gatcatatga aacaacatga ctttttcaag tctgccatgc
     3961 cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac tacaagacca
     4021 gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta aaaggtattg
     4081 attttaaaga agatggtaac attttaggtc acaaattgga atacaactat aactctcaca
     4141 atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc aaaattagac
     4201 acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat actccaattg
     4261 gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct gccttatcca
     4321 aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact gctgctggta
     4381 ttacccatgg tatggatgaa ttgtacaaat aactcgagta agcttggtac cgcggctagc
     4441 taagatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt
     4501 atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt
     4561 ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt
     4621 tgggacgctc gaagatccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4681 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4741 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4801 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4861 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4921 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4981 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5041 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5101 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5161 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5221 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5281 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5341 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5401 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5461 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5521 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5581 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5641 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5701 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5761 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5821 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5881 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5941 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6001 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6061 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6121 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6181 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6241 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6301 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6361 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6421 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6481 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6541 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6601 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6661 cctataaaaa taggcgtatc acgaggccct ttcgtc
//
