LOCUS       sequence-60873-e        6393 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 6393)
  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 6393)
  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-G1GZmbh
FEATURES             Location/Qualifiers
     source          1..6393
                     /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        317..504
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             505..1167
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     rep_origin      complement(1427..1882)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1564..1585)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1776..1795
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1965..2130)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2051..2075
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    2385..2401
                     /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        2532..2973
                     /label=TPGI promoter
                     /note="S. cerevisiae GAL1 promoter modified to contain two 
                     copies of the tet operator (Ellis et al., 2009)"
     protein_bind    2532..2649
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     protein_bind    2857..2875
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2878..2896
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2944..2967
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             2997..3710
                     /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"
     CDS             3735..4109
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      4142..4331
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4403..4420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4574..5162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4654..4673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5333..6124,6125..6193))
                     /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     5956..5975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6194..6298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6366..6384
                     /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 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt
      241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta
      301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat
      361 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat
      421 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag
      481 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca
      541 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac
      601 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg
      661 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat
      721 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc
      781 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt
      841 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg
      901 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta
      961 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga
     1021 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg
     1081 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt
     1141 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat
     1201 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta
     1261 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg
     1321 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg
     1381 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     1441 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1501 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     1561 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     1621 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     1681 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     1741 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     1801 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     1861 aatgcgccgc tacagggcgc gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa
     1921 gggcgatcgg tgcgggcctc ttcgctatta cgccagctga attggagcga cctcatgcta
     1981 tacctgagaa agcaacctga cctacaggaa agagttactc aagaataaga attttcgttt
     2041 taaaacctaa gagtcacttt aaaatttgta tacacttatt ttttttataa cttatttaat
     2101 aataaaaatc ataaatcata agaaattcgc ttatttagaa gtgtcaacaa cgtatctacc
     2161 aacgatttga cccttttcca tcttttcgta aatttctggc aaggtagaca agccgacaac
     2221 cttgattgga gacttgacca aacctctggc gaagaattgt taattaagag ctcttaacta
     2281 gtgcggccgc cctttagtga gggttgaatt cgaattttca aaaattctta cttttttttt
     2341 ggatggacgc aaagaagttt aataatcata ttacatgtgt gaaattgtta tccgctcaca
     2401 attccacaca acatatctaa tcttacttat atgttgtgga aatgtaaaga gccccattat
     2461 cttagcctaa aaaaaccttc tctttggaac tttcagtaat acgcttaact gctcattgct
     2521 atattgaagt acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc
     2581 ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc
     2641 actgctccga acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat
     2701 tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat
     2761 gataatgcga ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt
     2821 tttgatctat taacagatat ataaatgcaa aaactgtccc tatcagtgat agagatctcc
     2881 ctatcagtga tagagatatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt
     2941 gttaatatac ctctatactt taacgtcaag gagaaaaaac cccggatcct attaaaatgt
     3001 ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg
     3061 ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat
     3121 tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca accttagtca
     3181 ctactttcgg ttatggtgtt caatgttttg cgagataccc agatcatatg aaacaacatg
     3241 actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag
     3301 atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata
     3361 gaatcgaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg
     3421 aatacaacta taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca
     3481 aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt
     3541 atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat
     3601 ccactcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag
     3661 aatttgttac tgctgctggt attacccatg gtatggatga attgtacaaa ggtgacggtg
     3721 ctggtttaat taacatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     3781 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     3841 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     3901 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     3961 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
     4021 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
     4081 tgcacttcgt ggccgaggag caggactgac tcgagtaagc ttggtaccgc ggctagctaa
     4141 gatccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt
     4201 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc
     4261 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg
     4321 gacgctcgaa gatccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4981 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     5041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6301 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6361 ataaaaatag gcgtatcacg aggccctttc gtc
//
