LOCUS       sequence-246625-        7992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast plasmid overexpressing ADH2 from Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7992)
  AUTHORS   Liu Y, Bai C, Liu Q, Xu Q, Qian Z, Peng Q, Yu J, Xu M, Zhou X, Zhang
            Y, Cai M
  TITLE     Engineered ethanol-driven biosynthetic system for improving 
            production of acetyl-CoA derived drugs in Crabtree-negative yeast.
  JOURNAL   Metab Eng. 2019 Jul;54:275-284. doi: 10.1016/j.ymben.2019.05.001. 
            Epub 2019 May 9.
  PUBMED    31077813
REFERENCE   2  (bases 1 to 7992)
  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"; journalName: "Metab 
            Eng."; date: "2019-07"; volume: "54:275-284. doi"; pages: " 
            10.1016/j.ymben.2019.05.001. Epub 2019 May 9"
COMMENT     Sequence Label: pKdH_P-GAP-ScADH2
FEATURES             Location/Qualifiers
     source          1..7992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        116..1051
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     promoter        1060..1536
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2581..2598
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      2695..2941
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(3766..4581)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3766..4581)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4489..4508
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4573..4592
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4746..4765)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     misc_feature    4960..5708
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     primer_bind     complement(5743..5765)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5851..5991
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5906..5925
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6006..6023)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6177..6765)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6257..6276)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6936..7727,7728..7796))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7559..7578
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7797..7901)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7969..7987
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtcttcaag aattaattct catgtttgac agcttatcat cgataagctg actcatgttg
       61 gtattgtgaa atagacgcag atcgggaaca ctgaaaaata acagttatta ttcgagatct
      121 aacatccaaa gacgaaaggt tgaatgaaac ctttttgcca tccgacatcc acaggtccat
      181 tctcacacat aagtgccaaa cgcaacagga ggggatacac tagcagcaga ccgttgcaaa
      241 cgcaggacct ccactcctct tctcctcaac acccactttt gccatcgaaa aaccagccca
      301 gttattgggc ttgattggag ctcgctcatt ccaattcctt ctattaggct actaacacca
      361 tgactttatt agcctgtcta tcctggcccc cctggcgagg ttcatgtttg tttatttccg
      421 aatgcaacaa gctccgcatt acacccgaac atcactccag atgagggctt tctgagtgtg
      481 gggtcaaata gtttcatgtt ccccaaatgg cccaaaactg acagtttaaa cgctgtcttg
      541 gaacctaata tgacaaaagc gtgatctcat ccaagatgaa ctaagtttgg ttcgttgaaa
      601 tgctaacggc cagttggtca aaaagaaact tccaaaagtc ggcataccgt ttgtcttgtt
      661 tggtattgat tgacgaatgc tcaaaaataa tctcattaat gcttagcgca gtctctctat
      721 cgcttctgaa ccccggtgca cctgtgccga aacgcaaatg gggaaacacc cgctttttgg
      781 atgattatgc attgtctcca cattgtatgc ttccaagatt ctggtgggaa tactgctgat
      841 agcctaacgt tcatgatcaa aatttaactg ttctaacccc tacttgacag caatatataa
      901 acagaaggaa gctgccctgt cttaaacctt tttttttatc atcattatta gcttactttc
      961 ataattgcga ctggttccaa ttgacaagct tttgatttta acgactttta acgacaactt
     1021 gagaagatca aaaaacaact aattattcga agactagtct ttttgtagaa atgtcttggt
     1081 gtcctcgtcc aatcaggtag ccatctctga aatatctggc tccgttgcaa ctccgaacga
     1141 cctgctggca acgtaaaatt ctccggggta aaacttaaat gtggagtaat ggaaccagaa
     1201 acgtctcttc ccttctctct ccttccaccg cccgttaccg tcgctaggaa attttactct
     1261 gctggagagc ttcttctacg gcccccttgc agcaatgctc ttcccagcat tacgttgcgg
     1321 gtaaaacgga ggtcgtgtac ccgacctagc agcccaggga tggaaaagtc ccggccgtcg
     1381 ctggcaataa tagcgggcgg acgcatgtca tgagattatt ggaaaccacc agaatcgaat
     1441 ataaaaggcg aacacctttc ccaattttgg tttctcctga cccaaagact ttaaatttaa
     1501 tttatttgtc cctatttcaa tcaattgaac aactatatgt ctattccaga aactcaaaaa
     1561 gccattatct tctacgaatc caacggcaag ttggagcata aggatatccc agttccaaag
     1621 ccaaagccca acgaattgtt aatcaacgtc aagtactctg gtgtctgcca caccgatttg
     1681 cacgcttggc atggtgactg gccattgcca actaagttac cattagttgg tggtcacgaa
     1741 ggtgccggtg tcgttgtcgg catgggtgaa aacgttaagg gctggaagat cggtgactac
     1801 gccggtatca aatggttgaa cggttcttgt atggcctgtg aatactgtga attgggtaac
     1861 gaatccaact gtcctcacgc tgacttgtct ggttacaccc acgacggttc tttccaagaa
     1921 tacgctaccg ctgacgctgt tcaagccgct cacattcctc aaggtactga cttggctgaa
     1981 gtcgcgccaa tcttgtgtgc tggtatcacc gtatacaagg ctttgaagtc tgccaacttg
     2041 agagcaggcc actgggcggc catttctggt gctgctggtg gtctaggttc tttggctgtt
     2101 caatatgcta aggcgatggg ttacagagtc ttaggtattg atggtggtcc aggaaaggaa
     2161 gaattgttta cctcgctcgg tggtgaagta ttcatcgact tcaccaaaga gaaggacatt
     2221 gttagcgcag tcgttaaggc taccaacggc ggtgcccacg gtatcatcaa tgtttccgtt
     2281 tccgaagccg ctatcgaagc ttctaccaga tactgtaggg cgaacggtac tgttgtcttg
     2341 gttggtttgc cagccggtgc aaagtgctcc tctgatgtct tcaaccacgt tgtcaagtct
     2401 atctccattg tcggctctta cgtggggaac agagctgata ccagagaagc cttagatttc
     2461 tttgccagag gtctagtcaa gtctccaata aaggtagttg gcttatccag tttaccagaa
     2521 atttacgaaa agatggagaa gggccaaatt gctggtagat acgttgttga cacttctaaa
     2581 catcatcatc atcatcatta acctagggcg gccgcgaatt aattcgcctt agacatgact
     2641 gttcctcagt tcaagttggg cacttacgag aagaccggtc ttgctagatt ctaatcaaga
     2701 ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt
     2761 gtaacctata tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc
     2821 ctgatcagcc tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc
     2881 gagtttgatg tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag
     2941 acgttcgttt gtgcaagctt atcgataagc tttaatgcgg tagtttatca cagttaaatt
     3001 gctaacgcag tcaggcaccg tgtatgaaat ctaacaatgc gctcatcgtc atcctcggca
     3061 ccgtcaccct ggatgctgta ggcataggct tggttatgcc ggtactgccg ggcctcttgc
     3121 gggatatcgt ccattccgac agcatcgcca gtcactatgg cgtgctgcta gcgctatatg
     3181 cgttgatgca atttctatgc gcacccgttc tcggagcact gtccgaccgc tttggccgcc
     3241 gcccagtcct gctcgcttcg ctacttggag ccactatcga ctacgcgatc atggcgacca
     3301 cacccgtcct gtggatctat cgaatctaaa tgtaagttaa aatctctaaa taaacttgaa
     3361 gtcggacagt gagtgtagtc ttgagaaatt ctgaagccgt atttttatta tcagtgagtc
     3421 agtcatcagg agatcctcta cgccggacgc atcgtggccg acctgcaggg gggggggggc
     3481 gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga atcgccccat
     3541 catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag gtggaccagt
     3601 tggtgatttt gaacttttgc tttgccacgg aacggtctgc gttgtcggga agatgcgtga
     3661 tctgatcctt caactcagca aaagttcgat ttattcaaca aagccgccgt cccgtcaagt
     3721 cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc
     3781 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     3841 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     3901 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     3961 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     4021 tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     4081 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     4141 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     4201 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     4261 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     4321 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     4381 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     4441 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     4501 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt
     4561 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     4621 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     4681 aacgtggctt tccccccccc ccctgcaggt cggcatcacc ggcgccacag gtgcggttgc
     4741 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     4801 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     4861 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     4921 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagtatcta tgattggaag
     4981 tatgggaatg gtgatacccg cattcttcag tgtcttgagg tctcctatca gattatgccc
     5041 aactaaagca accggaggag gagatttcat ggtaaatttc tctgactttt ggtcatcagt
     5101 agactcgaac tgtgagacta tctcggttat gacagcagaa atgtccttct tggagacagt
     5161 aaatgaagtc ccaccaataa agaaatcctt gttatcagga acaaacttct tgtttcgaac
     5221 tttttcggtg ccttgaacta taaaatgtag agtggatatg tcgggtagga atggagcggg
     5281 caaatgctta ccttctggac cttcaagagg tatgtagggt ttgtagatac tgatgccaac
     5341 ttcagtgaca acgttgctat ttcgttcaaa ccattccgaa tccagagaaa tcaaagttgt
     5401 ttgtctacta ttgatccaag ccagtgcggt cttgaaactg acaatagtgt gctcgtgttt
     5461 tgaggtcatc tttgtatgaa taaatctagt ctttgatcta aataatcttg acgagccaga
     5521 cgataatacc aatctaaact ctttaaacgt taaaggacaa gtatgtctgc ctgtattaaa
     5581 ccccaaatca gctcgtagtc tgatcctcat caacttgagg ggcactatct tgttttagag
     5641 aaatttgcgg agatgcgata tcgagaaaaa ggtacgctga ttttaaacgt gaaatttatc
     5701 tcatgatctc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     5761 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     5821 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     5881 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5941 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     6001 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     6061 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     6121 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     6181 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     6241 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     6301 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     6361 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     6421 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     6481 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     6541 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     6601 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     6661 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     6721 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     6781 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6841 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     6901 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     6961 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     7021 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     7081 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     7141 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     7201 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     7261 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     7321 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     7381 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     7441 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     7501 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     7561 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     7621 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     7681 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     7741 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     7801 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     7861 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     7921 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     7981 acgaggccct tt
//
