LOCUS       sequence-248141-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  vector for replacing the promoter of FAS1 coding gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  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 6679)
  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: pAG_P-HXT1-FAS1up
FEATURES             Location/Qualifiers
     source          1..6679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..30)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(184..772)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(264..283)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(943..1734,1735..1803))
                     /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     1566..1585
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1804..1908)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1976..1994
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2032..2054)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2154..2173
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2254..2272
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2254..2271
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             4521..4538
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4618..4864
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     gene            4904..6479
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        4904..5247
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             5248..6276
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             5248..6276
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      6282..6479
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(6586..6605)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6587..6605)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
       61 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      121 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      181 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      241 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      301 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      361 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      421 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      481 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      541 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      601 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      661 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      721 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      781 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
      841 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
      901 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      961 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1021 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1081 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1141 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1201 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1261 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     1321 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     1381 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     1441 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     1501 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     1561 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     1621 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     1681 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     1741 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     1801 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1861 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1921 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     1981 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2041 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2101 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2161 agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa
     2221 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaacgcggcc
     2281 gccagctgaa gcttcgtacg ctgcaggtcg acgggtaccc aattgattaa gttcagtgaa
     2341 atttcaaacc gctatacaca acaggacaac tttgagttta gaaaaatccg atgtagtgta
     2401 acggctagca cggtccgctt tcaccgggca gacccgggtt cgactcccgg catcggagtt
     2461 tatttttcca tttcgttctt tagagtattc tcctcagcat gcccccctga atttttcctt
     2521 ttttccatgt gtcccatttt tccacttttt ttacagtttt cctcgtgatg ttaattggct
     2581 acacaaaagc tgccacacga aaccttaatc acgaaaaact atacagcctt cactaatccg
     2641 tagccccata atatgttgtc cacgtgctgt tgggtactac ctgtagactc tcatacccca
     2701 ctccgtcttt ctccaacaat taacgcagta ccgagattta tcagcagact caaattgggc
     2761 aaactctgta tttttccttg cccgcataat ttatgggtct caggcctcca cgtttcctgt
     2821 ttacttgaag aatattggct gcggaaaaag tggtaaggac aacccccttt taattggatc
     2881 cagtttttcc gaaatgttcc gatccgtacg tcatctccga agccgtacat tttcactcaa
     2941 tctacgtagc tttggactca gcgctcctgg aattgccagg acagtttact tgagttgata
     3001 ttcccttgta gattgtgtgc ttctttttcc aaaatttgag gcttcgtttg aaaagtggaa
     3061 tctggtcgct agatcacttc atgcctattt ttcacggaaa aataagtggt actatgcacc
     3121 ccttaaacct aaagaaaaac ggaaaaatta ccccaaaacc tggtgatgtt tttcgcccct
     3181 ttctttttat ccgagttttt cttttttctt gtctgccaaa ttcctctcct gaccttagcg
     3241 tccccggaaa aaattaacta cttaaggacc gaatgagccc cagcttttcc ccttctcttc
     3301 attattcccc ataatataat atgagtgcta catccggagt tgtcaacagg ccattggtgt
     3361 taaaccatgg ctcaatcgag tcaaccatct taatcccaac cacagaatat catttctacc
     3421 aaacgttact ggaagggttc aggaagtcat tgccacaagt taccgaggga tttgctgacg
     3481 acgacgagcc gagctccaag gcagaacttc tgatgaagtt tttgggttac attgtccaat
     3541 ctggagtctc caatcagcaa gagcaattag ctgctgcaaa gttggttttg aatgagttcg
     3601 agtcccgttt ccttcaagga ttgaacttgc actcttatgc tgccattttg cttaagagcg
     3661 agacctttcc cacaacatta ttgaaaatca aagaaaactt aatcaagaac tactacctgg
     3721 gaagagccct tgtctacctg ccaggtcagc gtggccttgt ataccctccc tcggcattac
     3781 ttaatgctgg aaagtccggc tccgctcaga tctacgccat atttggaggt caaggtaaca
     3841 ccgatgacta tttcgaagaa cttagagata tttaccacat ctaccaaggt ctcgtgtcag
     3901 acttcgttac caaggcccag ttgaagttac aagagctcat cagaaccacc cctgaaactg
     3961 acagaatata tacacagggt ctagacctga tcaattggct ggaaaacaag gacaagactc
     4021 ctgaccaaca acaattgcta tccattccaa tgtcatgtcc tctaatttgt gttatccaac
     4081 tgtgtcatta cattgtcact tgccgcatct taggtattac acctggtcaa ctcagagatt
     4141 ccttgaaagg tactacaggt cactcccaag gtctggtcac cgctgtggtc gtttcttctg
     4201 ctgattcctg ggaatccttt gagaaattgg cgcttcaggc agttgaattc atgttctaca
     4261 ttggtgttag aggactacaa acctatccaa acacctcttt acctccatcc atcgtccaag
     4321 attccgaaga aaatgccgag ggaactcctt ctcccatgct ttcagttaga gacctttcct
     4381 atgaccaatt ggtcaagttt gtcaacgaaa cgaaccagca cttgcctgag gccaaacata
     4441 tcgacatctc tttaataaat gggcccagaa atgtcgtctt gacaggccct cctcaatcac
     4501 tttacggttt gaatctgaac catcatcatc atcatcattg agtttgtagc cttagacatg
     4561 actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca
     4621 agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta
     4681 tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg
     4741 ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca
     4801 ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt
     4861 gagagtttag cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa
     4921 taccctcctt gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta
     4981 catttagccc atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac
     5041 ggcgcgaagc aaaaattacg gctcctcgct gcggacctgc gagcagggaa acgctcccct
     5101 cacagacgcg ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga
     5161 tttgccactg aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc
     5221 tcacatcaca tccgaacata aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct
     5281 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     5341 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     5401 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     5461 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     5521 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     5581 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     5641 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     5701 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     5761 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     5821 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     5881 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     5941 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     6001 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     6061 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     6121 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     6181 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     6241 cgacgcccca gcactcgtcc gagggcaaag gaataatcag tactgacaat aaaaagattc
     6301 ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat
     6361 caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt
     6421 taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc
     6481 tgtcgattcg atactaacgc cgccatccag tgtcgaaaac gagctcgaat tcatcgatga
     6541 tatcagatcc actagtggcc tatgcggccg cggatctgcc ggtctcccta tagtgagtcg
     6601 tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag
     6661 gcggtttgcg tattgggcg
//
