LOCUS       sequence-65290-e        5998 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 5998)
  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 5998)
  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: pXP812
FEATURES             Location/Qualifiers
     source          1..5998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      734..981
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(750..768)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    complement(1027..1060)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1078..1294
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             1295..2098
                     /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(2177..2210)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      2233..3575
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3196..3243)
                     /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(3595..3611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3595..3611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3608..3630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3619..3635
                     /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(3643..3649,3650..3667,3668..3673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3688..3709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3826..3843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3997..4585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4077..4096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4756..5547,5548..5616))
                     /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     5379..5398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5617..5721)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5789..5807
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5845..5867)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5967..5986
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 catatgaggc atttgcaaga attactcgtg agtaaggaaa gagtgaggaa ctatcgcata
       61 cctgcattta aagatgccga tttgggcgcg aatcctttat tttggcttca ccctcatact
      121 attatcaggg ccagaaaaag gaagtgtttc cctccttctt gaattgatgt taccctcata
      181 aagcacgtgg cctcttatcg agaaagaaat taccgtcgct cgtgatttgt ttgcaaaaag
      241 aacaaaactg aaaaaaccca gacacgctcg acttcctgtc ttcctattga ttgcagcttc
      301 caatttcgtc acacaacaag gtcctagcga cggctcacag gttttgtaac aagcaatcga
      361 aggttctgga atggcgggaa agggtttagt accacatgct atgatgccca ctgtgatctc
      421 cagagcaaag ttcgttcgat cgtactgtta ctctctctct ttcaaacaga attgtccgaa
      481 tcgtgtgaca acaacagcct gttctcacac actcttttct tctaaccaag ggggtggttt
      541 agtttagtag aacctcgtga aacttacatt tacatatata taaacttgca taaattggtc
      601 aatgcaagaa atacatattt ggtcttttct aattcgtagt ttttcaagtt cttagatgct
      661 ttctttttct cttttttaca gatcatcaag gaagtaatta tctacttttt acaacaaata
      721 tactagtctc gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca
      781 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
      841 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
      901 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
      961 cgctcgaagg ctttaatttg cggccaagct tgcatgcctg caggtcgact ctagaggatc
     1021 cccgggataa cttcgtatag catacattat acgaagttat cagggtccat aaagcttttc
     1081 aattcatctt tttttttttt gttctttttt ttgattccgg tttctttgaa atttttttga
     1141 ttcggtaatc tccgagcaga aggaagaacg aaggaaggag cacagactta gattggtata
     1201 tatacgcata tgtggtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca
     1261 cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg
     1321 tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca
     1381 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga
     1441 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc
     1501 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt
     1561 cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt
     1621 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat
     1681 tgttagcggt ttgaagcagg cggcggaaga agtaacaaag gaacctagag gccttttgat
     1741 gttagcagaa ttgtcatgca agggctccct agctactgga gaatatacta agggtactgt
     1801 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg
     1861 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa
     1921 gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta caggatctga
     1981 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga
     2041 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa
     2101 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt
     2161 atatcagtta ttacccataa cttcgtatag catacattat acgaagttat cccgggtacc
     2221 gagctcgaat tcaacgaagc atctgtgctt cattttgtag aacaaaaatg caacgcgaga
     2281 gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg
     2341 aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa aaatgcaacg
     2401 cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa cagaaatgca
     2461 acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc tacaaaaatg
     2521 catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt ctcctttgtg
     2581 cgctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa ggttagaaga
     2641 aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac ttcccgcgtt
     2701 tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc cccgattata
     2761 ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg ttgatgattc
     2821 ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata ctacgtatag
     2881 gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct tactacaatt
     2941 tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc gagtttagat
     3001 gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc acagagatat
     3061 atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat attttagtag
     3121 ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag cgcttttggt
     3181 tttcaaaagc gctctgaagt tcctatactt tctagagaat aggaacttcg gaataggaac
     3241 ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc gcacatacag
     3301 ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat acatgagaag
     3361 aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt tatgtaggat
     3421 gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta tcgtatgctt
     3481 ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg attagtctca
     3541 tccttcaatg ctatcatttc ctttgatatt ggatcatacg aattcgtaat catggtcata
     3601 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3661 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     3721 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3781 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3841 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3901 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3961 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4021 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4081 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4141 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4201 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4261 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4321 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4381 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4441 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4501 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4561 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4621 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4681 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4741 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4801 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4861 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4921 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4981 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5041 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5101 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5161 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5221 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5281 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5341 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5401 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5461 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5521 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5581 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5641 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5701 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     5761 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     5821 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     5881 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     5941 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcac
//
