LOCUS       sequence-65294-e        5589 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 5589)
  AUTHORS   Shen MW, Fang F, Sandmeyer S, Da Silva NA
  TITLE     Development and characterization of a vector set with regulated 
            promoters for systematic metabolic engineering in Saccharomyces 
            cerevisiae.
  JOURNAL   Yeast. 2012 Dec;29(12):495-503. doi: 10.1002/yea.2930. Epub 2012 Nov
            19.
  PUBMED    23166051
REFERENCE   2  (bases 1 to 5589)
  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: "10.1002/yea.2930"; 
            journalName: "Yeast"; date: "2012-12"; volume: "29"; issue: "12"; 
            pages: "495-503"
COMMENT     Sequence Label: pXP832
FEATURES             Location/Qualifiers
     source          1..5589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        22..256
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     terminator      325..572
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(341..359)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    complement(618..651)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        669..885
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             886..1689
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLATGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     protein_bind    complement(1768..1801)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      1824..3166
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(2787..2834)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(3186..3202)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3186..3202)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3199..3221)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3210..3226
                     /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        complement(join(3234..3240,3241..3258,3259..3264))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3279..3300
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3417..3434)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3588..4176)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3668..3687)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4347..5138,5139..5207))
                     /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     4970..4989
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5208..5312)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5380..5398
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5436..5458)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5558..5577
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catatgatct tagccttgtt actagttaga aaaagacatt tttgctgtca gtcactgtca
       61 agagattctt ttgctggcat ttcttctaga agcaaaaaga gcgatgcgtc ttttccgctg
      121 aaccgttcca gcaaaaaaga ctaccaacgc aatatggatt gtcagaatca tataaaagag
      181 aagcaaataa ctccttgtct tgtatcaatt gcattataat atcttcttgt tagtgcaata
      241 tcatatagaa gtcatcgaaa tagatattaa gaaaaacaaa ctgtacaatc aatcaatcaa
      301 tcatcacata aacctaggct cgagtcatgt aattagttat gtcacgctta cattcacgcc
      361 ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc
      421 ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc
      481 ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga
      541 aggttttggg acgctcgaag gctttaattt gcggccaagc ttgcatgcct gcaggtcgac
      601 tctagaggat ccccgggata acttcgtata gcatacatta tacgaagtta tcagggtcca
      661 taaagctttt caattcatct tttttttttt tgttcttttt tttgattccg gtttctttga
      721 aatttttttg attcggtaat ctccgagcag aaggaagaac gaaggaagga gcacagactt
      781 agattggtat atatacgcat atgtggtgtt gaagaaacat gaaattgccc agtattctta
      841 acccaactgc acagaacaaa aacctgcagg aaacgaagat aaatcatgtc gaaagctaca
      901 tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg
      961 cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg
     1021 gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg
     1081 actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat
     1141 tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac
     1201 tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg
     1261 ggcccaggta ttgttagcgg tttgaagcag gcggcggaag aagtaacaaa ggaacctaga
     1321 ggccttttga tgttagcaga attgtcatgc aagggctccc tagctactgg agaatatact
     1381 aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa
     1441 agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt
     1501 ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct
     1561 acaggatctg acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag
     1621 gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag
     1681 caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct
     1741 tcaatttaat tatatcagtt attacccata acttcgtata gcatacatta tacgaagtta
     1801 tcccgggtac cgagctcgaa ttcaacgaag catctgtgct tcattttgta gaacaaaaat
     1861 gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga
     1921 aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca
     1981 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga
     2041 acagaaatgc aacgcgagag cgctatttta ccaacaaaga atctatactt cttttttgtt
     2101 ctacaaaaat gcatcccgag agcgctattt ttctaacaaa gcatcttaga ttactttttt
     2161 tctcctttgt gcgctctata atgcagtctc ttgataactt tttgcactgt aggtccgtta
     2221 aggttagaag aaggctactt tggtgtctat tttctcttcc ataaaaaaag cctgactcca
     2281 cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat
     2341 ccccgattat attctatacc gatgtggatt gcgcatactt tgtgaacaga aagtgatagc
     2401 gttgatgatt cttcattggt cagaaaatta tgaacggttt cttctatttt gtctctatat
     2461 actacgtata ggaaatgttt acattttcgt attgttttcg attcactcta tgaatagttc
     2521 ttactacaat ttttttgtct aaagagtaat actagagata aacataaaaa atgtagaggt
     2581 cgagtttaga tgcaagttca aggagcgaaa ggtggatggg taggttatat agggatatag
     2641 cacagagata tatagcaaag agatactttt gagcaatgtt tgtggaagcg gtattcgcaa
     2701 tattttagta gctcgttaca gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga
     2761 gcgcttttgg ttttcaaaag cgctctgaag ttcctatact ttctagagaa taggaacttc
     2821 ggaataggaa cttcaaagcg tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg
     2881 cgcacataca gctcactgtt cacgtcgcac ctatatctgc gtgttgcctg tatatatata
     2941 tacatgagaa gaacggcata gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat
     3001 ttatgtagga tgaaaggtag tctagtacct cctgtgatat tatcccattc catgcggggt
     3061 atcgtatgct tccttcagca ctacccttta gctgttctat atgctgccac tcctcaattg
     3121 gattagtctc atccttcaat gctatcattt cctttgatat tggatcatac gaattcgtaa
     3181 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3241 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3301 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3361 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3421 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3481 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3541 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3601 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3661 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3721 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3781 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     3841 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3901 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3961 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4021 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4081 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4141 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4201 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4261 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4321 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4381 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4441 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4501 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4561 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4621 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4681 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4741 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4801 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     4861 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4921 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4981 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5041 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5101 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5161 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5221 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5281 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5341 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     5401 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     5461 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     5521 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     5581 agagtgcac
//
