LOCUS       sequence-246626-        9162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  yeast plasmid overexpressing ACS1 from Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9162)
  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 9162)
  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-ScACS1*
FEATURES             Location/Qualifiers
     source          1..9162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        134..1069
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     promoter        1078..1554
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             3724..3753
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3769..3786
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3883..4129
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(4954..5769)
                     /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(4954..5769)
                     /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     5677..5696
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5761..5780
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5934..5953)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     misc_feature    6148..6896
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     primer_bind     complement(6931..6953)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    7039..7179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     7094..7113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(7194..7211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7365..7953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7445..7464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8124..8915,8916..8984))
                     /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     8747..8766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8985..9089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(9157..9162,1..13)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtatcacga ggccctttcg tcttcaagaa ttaattctca tgtttgacag cttatcatcg
       61 ataagctgac tcatgttggt attgtgaaat agacgcagat cgggaacact gaaaaataac
      121 agttattatt cgagatctaa catccaaaga cgaaaggttg aatgaaacct ttttgccatc
      181 cgacatccac aggtccattc tcacacataa gtgccaaacg caacaggagg ggatacacta
      241 gcagcagacc gttgcaaacg caggacctcc actcctcttc tcctcaacac ccacttttgc
      301 catcgaaaaa ccagcccagt tattgggctt gattggagct cgctcattcc aattccttct
      361 attaggctac taacaccatg actttattag cctgtctatc ctggcccccc tggcgaggtt
      421 catgtttgtt tatttccgaa tgcaacaagc tccgcattac acccgaacat cactccagat
      481 gagggctttc tgagtgtggg gtcaaatagt ttcatgttcc ccaaatggcc caaaactgac
      541 agtttaaacg ctgtcttgga acctaatatg acaaaagcgt gatctcatcc aagatgaact
      601 aagtttggtt cgttgaaatg ctaacggcca gttggtcaaa aagaaacttc caaaagtcgg
      661 cataccgttt gtcttgtttg gtattgattg acgaatgctc aaaaataatc tcattaatgc
      721 ttagcgcagt ctctctatcg cttctgaacc ccggtgcacc tgtgccgaaa cgcaaatggg
      781 gaaacacccg ctttttggat gattatgcat tgtctccaca ttgtatgctt ccaagattct
      841 ggtgggaata ctgctgatag cctaacgttc atgatcaaaa tttaactgtt ctaaccccta
      901 cttgacagca atatataaac agaaggaagc tgccctgtct taaacctttt tttttatcat
      961 cattattagc ttactttcat aattgcgact ggttccaatt gacaagcttt tgattttaac
     1021 gacttttaac gacaacttga gaagatcaaa aaacaactaa ttattcgaag actagtcttt
     1081 ttgtagaaat gtcttggtgt cctcgtccaa tcaggtagcc atctctgaaa tatctggctc
     1141 cgttgcaact ccgaacgacc tgctggcaac gtaaaattct ccggggtaaa acttaaatgt
     1201 ggagtaatgg aaccagaaac gtctcttccc ttctctctcc ttccaccgcc cgttaccgtc
     1261 cctaggaaat tttactctgc tggagagctt cttctacggc ccccttgcag caatgctctt
     1321 cccagcatta cgttgcgggt aaaacggagg tcgtgtaccc gacctagcag cccagggatg
     1381 gaaaagtccc ggccgtcgct ggcaataata gcgggcggac gcatgtcatg agattattgg
     1441 aaaccaccag aatcgaatat aaaaggcgaa cacctttccc aattttggtt tctcctgacc
     1501 caaagacttt aaatttaatt tatttgtccc tatttcaatc aattgaacaa ctatatgtcg
     1561 ccctctgccg tacaatcatc aaaactagaa gaacagtcaa gtgaaattga caagttgaaa
     1621 gcaaaaatgt cccagtctgc ctccactgcg cagcagaaga aggaacatga gtatgaacat
     1681 ttgacctcgg tcaagatcgt gccacaacgg cccatctcag atagactgca gcccgcaatt
     1741 gctacccact attctccaca cttggacggg ttgcaggact atcagcgctt gcacaaggag
     1801 tctattgaag accctgctaa gttcttcggt tctaaagcta cccaattttt aaactggtct
     1861 aagccattcg ataaggtgtt catcccagac tctaaaacgg gtaggccctc cttccagaac
     1921 aatgcatggt tcctcaacgg ccaattaaac gcctgttaca actgtgttga cagacatgcc
     1981 ttgaagaccc ctaacaagaa agccattatt ttcgaaggtg acgagcctgg ccaaggctat
     2041 tccattacct acaaggaact acttgaagaa gtttgtcaag tggcacaagt gctgacttac
     2101 tctatgggcg ttcgcaaggg cgatactgtt gccgtgtaca tgcctatggt cccagaagca
     2161 atcataacct tgttggccat ttcccgtatc ggcgccattc actccgtagt ctttgccggg
     2221 ttttcttcca actccttgag agatcgtatc aacgatgggg actctaaagt tgtcatcact
     2281 acagatgaat ccaacagagg tggtaaagtc attgagacta aaagaattgt tgatgacgcg
     2341 ctaagagaga ccccaggcgt gagacacgtc ttggtttata gaaagaccaa caatccatct
     2401 gttgctttcc atgcccccag agatttagat tgggcaacag aaaagaagaa atacaagacc
     2461 tactatccat gcacacccgt tgattctgag gatccattat tcttgttgta tacgtctggt
     2521 tctactggtg cccccaaggg tgttcaacat tctaccgcag gttacttgct gggagctttg
     2581 ttgaccatgc gctacacttt tgacactcac caagaagacg ttttcttcac agctggagac
     2641 attggctgga ttacaggcca cacttatgtg gtttatggtc ccttactata tggttgtgcc
     2701 actttggtct ttgaagggac tcctgcgtac ccaaattact cccgttattg ggatattatt
     2761 gatgaacaca aagtcaccca attttatgtt gccccaactg ctttgcgttt gttgaaaaga
     2821 gctggtgatt cctacatcga aaatcattcc ttaaaatctt tgcgttgctt gggttcggtc
     2881 ggtgaaccaa ttgctgctga agtttgggag tggtactctg aaaaaatagg taaaaatgaa
     2941 atccccattg tagacaccta ctggcaaaca gaatctggtt cgcatctggt caccccgctg
     3001 gctggtggtg tcacaccaat gaaaccgggt tctgcctcat tccccttctt cggtattgat
     3061 gcagttgttc ttgaccctaa cactggtgaa gaacttaata ccagccacgc agagggtgtc
     3121 cttgccgtca aagctgcatg gccatcattt gcaagaacta tttggaaaaa tcatgatagg
     3181 tatctagaca cttatttgaa cccttaccct ggctactatt tcactggtga tggtgctgca
     3241 aaggataagg atggttatat ctggattttg ggtcgtgtag acgatgtggt gaacgtctct
     3301 ggtcaccgtc tgtctaccgc tgaaattgag gctgctatta tcgaagatcc aattgtggcc
     3361 gagtgtgctg ttgtcggatt caacgatgac ttgactggtc aagcagttgc tgcatttgtg
     3421 gtgttgaaaa acaaatctaa ttggtccacc gcaacagatg atgaattaca agatatcaag
     3481 aagcatttgg tctttactgt tagaaaagac atcgggccat ttgccgcacc aaaattgatc
     3541 attttagtgg atgacttgcc caagacaaga tctggcaaaa ttatgagacg tattttaaga
     3601 aaaatcctag caggagaaag tgaccaacta ggcgacgttt ctacattgtc aaaccctggc
     3661 attgttagac atccaattga ttcggtcaag ttgcctcgag ccgcggcggc cgccagcttt
     3721 ctagaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat
     3781 catcattgac ctagggcggc cgcgaattaa ttcgccttag acatgactgt tcctcagttc
     3841 aagttgggca cttacgagaa gaccggtctt gctagattct aatcaagagg atgtcagaat
     3901 gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt aacctatata
     3961 gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct gatcagccta
     4021 tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt
     4081 tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac gttcgtttgt
     4141 gcaagcttat cgataagctt taatgcggta gtttatcaca gttaaattgc taacgcagtc
     4201 aggcaccgtg tatgaaatct aacaatgcgc tcatcgtcat cctcggcacc gtcaccctgg
     4261 atgctgtagg cataggcttg gttatgccgg tactgccggg cctcttgcgg gatatcgtcc
     4321 attccgacag catcgccagt cactatggcg tgctgctagc gctatatgcg ttgatgcaat
     4381 ttctatgcgc acccgttctc ggagcactgt ccgaccgctt tggccgccgc ccagtcctgc
     4441 tcgcttcgct acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt
     4501 ggatctatcg aatctaaatg taagttaaaa tctctaaata aacttgaagt cggacagtga
     4561 gtgtagtctt gagaaattct gaagccgtat ttttattatc agtgagtcag tcatcaggag
     4621 atcctctacg ccggacgcat cgtggccgac ctgcaggggg gggggggcgc tgaggtctgc
     4681 ctcgtgaaga aggtgttgct gactcatacc aggcctgaat cgccccatca tccagccaga
     4741 aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg gtgattttga
     4801 acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca
     4861 actcagcaaa agttcgattt attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct
     4921 ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg
     4981 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     5041 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     5101 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     5161 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt
     5221 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     5281 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     5341 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     5401 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     5461 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     5521 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     5581 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     5641 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     5701 taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat
     5761 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     5821 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc
     5881 cccccccccc ctgcaggtcg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
     5941 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
     6001 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
     6061 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
     6121 aatgcaggag tcgcataagg gagagcgtcg agtatctatg attggaagta tgggaatggt
     6181 gatacccgca ttcttcagtg tcttgaggtc tcctatcaga ttatgcccaa ctaaagcaac
     6241 cggaggagga gatttcatgg taaatttctc tgacttttgg tcatcagtag actcgaactg
     6301 tgagactatc tcggttatga cagcagaaat gtccttcttg gagacagtaa atgaagtccc
     6361 accaataaag aaatccttgt tatcaggaac aaacttcttg tttcgaactt tttcggtgcc
     6421 ttgaactata aaatgtagag tggatatgtc gggtaggaat ggagcgggca aatgcttacc
     6481 ttctggacct tcaagaggta tgtagggttt gtagatactg atgccaactt cagtgacaac
     6541 gttgctattt cgttcaaacc attccgaatc cagagaaatc aaagttgttt gtctactatt
     6601 gatccaagcc agtgcggtct tgaaactgac aatagtgtgc tcgtgttttg aggtcatctt
     6661 tgtatgaata aatctagtct ttgatctaaa taatcttgac gagccagacg ataataccaa
     6721 tctaaactct ttaaacgtta aaggacaagt atgtctgcct gtattaaacc ccaaatcagc
     6781 tcgtagtctg atcctcatca acttgagggg cactatcttg ttttagagaa atttgcggag
     6841 atgcgatatc gagaaaaagg tacgctgatt ttaaacgtga aatttatctc atgatctctg
     6901 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6961 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7021 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     7081 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     7141 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     7201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     7261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     7321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     7381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     7441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     7501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     7561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     7621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     7681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     7741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     7801 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     7861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     8041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     8101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     8161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     8221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     8281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     8341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     8401 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     8461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     8521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     8581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     8641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     8701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     8761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     8821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     8881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     9001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     9061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     9121 taagaaacca ttattatcat gacattaacc tataaaaata gg
//
