LOCUS       sequence-431852-        5984 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for constitutive expression of LDH at the 
            LEU2 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5984)
  AUTHORS   Choi B, Tafur Rangel A, Kerkhoven EJ, Nygard Y
  TITLE     Engineering of Saccharomyces cerevisiae for enhanced metabolic 
            robustness and L-lactic acid production from lignocellulosic 
            biomass.
  JOURNAL   Metab Eng. 2024 Jul;84:23-33. doi: 10.1016/j.ymben.2024.05.003. Epub
            2024 May 23.
  PUBMED    38788894
REFERENCE   2  (bases 1 to 5984)
  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: "2024-07"; volume: "84:23-33. doi"; pages: " 
            10.1016/j.ymben.2024.05.003. Epub 2024 May 23"
COMMENT     Sequence Label: YIplac128-TEF2p-LDH-CYC1t
FEATURES             Location/Qualifiers
     source          1..5984
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(142..147,148..165,166..172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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)."
     primer_bind     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      1729..1976
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1745..1763)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(1983..2000)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1983..1999)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1992..2014)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2208..2227)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2327..2349
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2490..3584)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(3585..3990)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(3992..4010)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4078..4182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4183..4251,4252..5043)
                     /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     complement(4401..4420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5214..5802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5703..5722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5956..5973
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
      241 atgcctgcag ctgcagccgc gacaaattac ccataaggtt gtttgtgacg gcgtcgtaca
      301 agagaacgtg ggaacttttt aggctcacca aaaaagaaag aaaaaatacg agttgctgac
      361 agaagcctca agaaaaaaaa aattcttctt cgactatgct ggaggcagag atgatcgagc
      421 cggtagttaa ctatatatag ctaaattggt tccatcacct tcttttctgg tgtcgctcct
      481 tctagtgcta tttctggctt ttcctatttt tttttttcca tttttctttc tctctttcta
      541 atatataaat tctcttgcat tttctatttt tctctctatc tattctactt gtttattccc
      601 ttcaaggttt ttttttaagg agtacttgtt tttagaatat acggtcaacg aactataatt
      661 aacctgcagc tgcaggtcga ctctagagca tggcgacact taaggatcag ttgatccaaa
      721 atctactgaa agaagaacac gtaccacaga acaagataac catcgtcggg gtcggtgccg
      781 tcggaatggc ctgcgcaata agtatcttaa tgaaagactt agcggacgaa gttgcactgg
      841 tggatgttat ggaggacaaa cttaagggcg aaatgatgga tctgcagcac ggatcattgt
      901 tcttacgtac gcccaaaatc gttagtggta aggactataa tgtaacagct aatagcaggt
      961 tggtaatcat tactgccgga gccaggcaac aagaaggcga atcaaggtta aatctggtac
     1021 agagaaatgt gaacattttt aagttcataa tcccaaatat cgttaagtat agccccaatt
     1081 gcaaacttct agtagtgtca aacccggtgg atatcctaac atacgtggcc tggaaaatct
     1141 cagggttccc gaaaaatcgt gtaattggat caggatgcaa cctggattcc gccagattcc
     1201 gttaccttat gggcgagagg ctgggtgtcc atcctctaag ttgccacgga tggattttag
     1261 gggagcatgg cgacagttca gtacccgtct ggagcggggt caacgtagct ggtgtcagtc
     1321 ttaagaattt gcacccggag ctgggtactg acgccgacaa ggaacaatgg aaagctgtgc
     1381 ataagcaagt agttgattcc gcttacgaag tgattaagtt aaagggttat acaagttggg
     1441 ccataggcct atccgtcgca gatttagcag agagcattat gaaaaacctt cgtcgtgtcc
     1501 atccgatcag cacaatgatt aaaggtttgt atgggattaa agaagacgtt ttcttgagtg
     1561 ttccttgcat actagggcaa aatggtatct cagatgtcgt caaagtgacc cttactcacg
     1621 aagaagaggc atgtcttaag aagtctgcag acaccctttg gggaattcag aaggaactac
     1681 agttctaacc cccgggtacc gagctcggcc ccttttcctt tgtcgatatc atgtaattag
     1741 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     1801 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     1861 cgttatttat atttcaaatt tttctttttt ttctgtacaa acgcgtgtac gcatgtaaca
     1921 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcgaat
     1981 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2041 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2101 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     2161 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     2221 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2281 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2341 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct accctatgaa
     2401 catattccat tttgtaattt cgtgtcgttt ctattatgaa tttcatttat aaagtttatg
     2461 tacaaatatc ataaaaaaag agaatctttt taagcaagga ttttcttaac ttcttcggcg
     2521 acagcatcac cgacttcggt ggtactgttg gaaccaccta aatcaccagt tctgatacct
     2581 gcatccaaaa cctttttaac tgcatcttca atggccttac cttcttcagg caagttcaat
     2641 gacaatttca acatcattgc agcagacaag atagtggcga tagggttgac cttattcttt
     2701 ggcaaatctg gagcagaacc gtggcatggt tcgtacaaac caaatgcggt gttcttgtct
     2761 ggcaaagagg ccaaggacgc agatggcaac aaacccaagg aacctgggat aacggaggct
     2821 tcatcggaga tgatatcacc aaacatgttg ctggtgatta taataccatt taggtgggtt
     2881 gggttcttaa ctaggatcat ggcggcagaa tcaatcaatt gatgttgaac cttcaatgta
     2941 ggaaattcgt tcttgatggt ttcctccaca gtttttctcc ataatcttga agaggccaaa
     3001 acattagctt tatccaagga ccaaataggc aatggtggct catgttgtag ggccatgaaa
     3061 gcggccattc ttgtgattct ttgcacttct ggaacggtgt attgttcact atcccaagcg
     3121 acaccatcac catcgtcttc ctttctctta ccaaagtaaa tacctcccac taattctctg
     3181 acaacaacga agtcagtacc tttagcaaat tgtggcttga ttggagataa gtctaaaaga
     3241 gagtcggatg caaagttaca tggtcttaag ttggcgtaca attgaagttc tttacggatt
     3301 tttagtaaac cttgttcagg tctaacacta cctgtacccc atttaggacc acccacagca
     3361 cctaacaaaa cggcatcagc cttcttggag gcttccagcg cctcatctgg aagtggaaca
     3421 cctgtagcat cgatagcagc accaccaatt aaatgatttt cgaaatcgaa cttgacattg
     3481 gaacgaacat cagaaatagc tttaagaacc ttaatggctt cggctgtgat ttcttgacca
     3541 acgtggtcac ctggcaaaac gacgatcttc ttaggggcag acattagaat ggtatatcct
     3601 tgaaatatat atatatattg ctgaaatgta aaaggtaaga aaagttagaa agtaagacga
     3661 ttgctaacca cctattggaa aaaacaatag gtccttaaat aatattgtca acttcaagta
     3721 ttgtgatgca agcatttagt catgaacgct tctctattct atatgaaaag ccggttccgg
     3781 cgctctcacc tttccttttt ctcccaattt ttcagttgaa aaaggtatat gcgtcaggcg
     3841 acctctgaaa ttaacaaaaa atttccagtc atcgaatttg attctgtgcg atagcgcccc
     3901 tgtgtgttct cgttatgttg aggaaaaaaa taatggttgc taagagattc gaactcttgc
     3961 atcttacgat acctgagtat tcccacagtt ggcctcgtga tacgcctatt tttataggtt
     4021 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4081 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4141 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4201 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4261 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4321 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4381 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4441 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4501 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4561 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     4621 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     4681 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     4741 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     4801 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     4861 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     4921 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     4981 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5041 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5101 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5161 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5221 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5281 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5341 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5401 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5461 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5521 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5581 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5641 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5701 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5761 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5821 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5881 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     5941 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaga
//
