LOCUS       sequence-359550-        9603 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for heterologous expression of a polyketide 
            synthase in S. cerevisiae. Carries and empty insertion site and the 
            Fusarium verticillioides 4´-phosphopantetheinyltransferase PPT1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9603)
  AUTHORS   Bejenari M, Nielsen L, Spedtsberg EML, Nielsen MR, Pedersen TB, 
            Sorensen JL
  TITLE     Yeast recombinational cloning for heterologous biosynthesis of 
            polyketides: a molecular microbiology laboratory module for 
            undergraduate students.
  JOURNAL   J Microbiol Biol Educ. 2023 Nov 27;24(3):e00242-22. doi: 
            10.1128/jmbe.00242-22. eCollection 2023 Dec.
  PUBMED    38108002
REFERENCE   2  (bases 1 to 9603)
  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: 
            "10.1128/jmbe.00242-22"; journalName: "J Microbiol Biol Educ"; date:
            "2023-11-27- 27"; volume: "24"; issue: "3"; pages: "e00242-22"
COMMENT     Sequence Label: pCOA-Leu-FvPPT1
FEATURES             Location/Qualifiers
     source          1..9603
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     396..415
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(909..2003)
                     /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(2004..2411)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2712..3167)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2849..2870)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3061..3080
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3246..3411)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3332..3356
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3559..3582)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     5043..5062
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5043..5061
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      6010..6199
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6271..6288)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6442..7030)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6522..6541)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7201..7992,7993..8061))
                     /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     7824..7843
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8062..8166)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      8193..9535
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(9156..9203)
                     /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     9576..9594
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gccaattatg ttatgtctga
       61 taaaaggatg taacataggc aagctgctcg tgagtgttga gtacgaacct tagatccaaa
      121 tcacccgcac cctacggata tacttgcttg aatatacagt agtatttgta ataagttcat
      181 aattaatatc ttaccaaatt aggtaatctg caaaagttca gactgtgaaa tgtaacattt
      241 tatatatcaa gctctattta atgcctcaca gtagttaaca taaagacagc tcccggagac
      301 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      361 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      421 agtgcaccat atcgactacg tcgttaaggc cgtttctgac agagtaaaat tcttgaggga
      481 actttcacca ttatgggaaa tggttcaaga aggtattgac ttaaactcca tcaaatggtc
      541 aggtcattga gtgtttttta tttgttgtat tttttttttt ttagagaaaa tcctccaata
      601 tataaattag gaatcatagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc
      661 ctcctccttg tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat
      721 tagtatcaat ttgcttacct gtattccttt acatcctcct ttttctcctt cttgataaat
      781 gtatgtagat tgcgtatata gtttcgtcta ccctatgaac atattccatt ttgtaatttc
      841 gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga
      901 gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg
      961 gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact
     1021 gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca
     1081 gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg
     1141 tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca
     1201 gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca
     1261 aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg
     1321 gcggcagaat caatcaattg atgttgaacc ttcaatgtag ggaattcgtt cttgatggtt
     1381 tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac
     1441 caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt
     1501 tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc
     1561 tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct
     1621 ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat
     1681 ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt
     1741 ctaacactac cggtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc
     1801 ttcttggagg cttccagcgc ctcatctgga agtggaacac ctgtagcatc gatagcagca
     1861 ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct
     1921 ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg
     1981 acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata tatatatatt
     2041 gctgaaatgt aaaaggtaag aaaagttaga aagtaagacg attgctaacc acctattgga
     2101 aaaaacaata ggtccttaaa taatattgtc aacttcaagt attgtgatgc aagcatttag
     2161 tcatgaacgc ttctctattc tatatgaaaa gccggttccg gcgctctcac ctttcctttt
     2221 tctcccaatt tttcagttga aaaaggtata tgcgtcaggc gacctctgaa attaacaaaa
     2281 aatttccagt catcgaattt gattctgtgc gatagcgccc ctgtgtgttc tcgttatgtt
     2341 gaggaaaaaa ataatggttg ctaagagatt cgaactcttg catcttacga tacctgagta
     2401 ttcccacagt taactgcggt caagatattt cttgaatcag gcgccttaga ccgctcggcc
     2461 aaacaaccaa ttacttgttg agaaatagag tataattatc ctataaatat aacgtttttg
     2521 aacacacatg aacaaggaag tacaggacaa ttgattttga agagaatgtg gattttgatg
     2581 taattgttgg gattccattt ttaataaggc aataatatta ggtatgtaga tatactagaa
     2641 gttctcctcg accggtcgat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     2701 accgcatcag gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     2761 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2821 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2881 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2941 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     3001 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     3061 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     3121 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcca ttcgccattc
     3181 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     3241 aattggagcg acctcatgct atacctgaga aagcaacctg acctacagga aagagttact
     3301 caagaataag aattttcgtt ttaaaaccta agagtcactt taaaatttgt atacacttat
     3361 tttttttata acttatttaa taataaaaat cataaatcat aagaaattcg cttatttaga
     3421 agtgtcaaca acgtatctac caacgatttg acccttttcc atcttttcgt aaatttctgg
     3481 caaggtagac aagccgacaa ccttgattgg agacttgacc aaacctctgg cgaagaattg
     3541 ttaattaaga gctcagatct tatcgtcgtc atccttgtaa tccatcgata ctagtgcggc
     3601 cgccctttag tgagggttga attcgaattt tgaatgattc ttatactcag aaggaaatgc
     3661 ttaacgattt cgggtgtgag ttgacaagga gagagagaaa agaagaggaa aggtaattcg
     3721 gggacggtgg tcttttatac ccttggctaa agtcccaacc acaaagcaaa aaaattttca
     3781 gtagtctatt ttgcgtccgg catgggttac ccggatggcc agacaaagaa actagtacaa
     3841 agtctgaaca agcgtagatt ccagactgca gtaccctacg cccttaacgg caagtgtggg
     3901 aaccggggga ggtttgatat gtggggtgaa gggggctctc gccggggttg ggcccgctac
     3961 tgggtcaatt tggggtcaat tggggcaatt ggggctgttt tttgggacac aaatacgccg
     4021 ccaacccggt ctctgagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat
     4081 gagcggcggc agattcgagc gtttccggtt tccgcggcgg gacgagagcc catgatgggg
     4141 gctcccacca ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc
     4201 atcgtggtta atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt
     4261 atgcagcgta caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg
     4321 cttaacaaca ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga
     4381 ggcgttttgg agcggcttgg cctggcgccc actcgcgaaa cgcacctagg accctttggc
     4441 acgccgaaat gtgccacttt tcagtctagt aacgccttac ctacgtcatt ccatgcatgc
     4501 atgtttgcgc cttttttccc ttgcccttga tcgccacaca gtacagtgca ctgtacagtg
     4561 gaggttttgg gggggtctta gatgggagct aaaagcggcc tagcggtaca ctagtgggat
     4621 tgtatggagt ggcatggagc ctgggtggag cctgacagga cgcacgaccg gctagcccgt
     4681 gacagacgat gggtggctcc tgttgtccac cgcgtacaaa tgtttgggcc aaagtcttgt
     4741 cagccttgct tgcgaaccta attcccaatt ttgtcacttc gcacccccat tgatcgagcc
     4801 ctaacccctg cccatcaggc aatccaatta agctcgcatt gtctgccttg tttagtttgg
     4861 ctcctgcccg tttcggcgtc cacttgcaca aacacaaaca agcattatat ataaggctcg
     4921 tctctccctc ccaaccacac tcactttttt gcccgtcttc ccttgctaac acaaaagtca
     4981 agaacacaaa caaccacccc aaccccctta cacacaagac atatctacag cacccggatc
     5041 cgtaatacga ctcactatag ggcccgggcg tcgacatgga acagaagttg atttccgaag
     5101 aagaccatgt cctcagcaca atcatcacca acagtaatac aatgggtaat agatacaaga
     5161 cctttatggc cttccgtttt gaagactaaa gatttgactt ctgctgcatc aagagctttg
     5221 tctttgttga cagaagaaga acaatcttca gttttgagat actaccatgt tagagatgca
     5281 aaattggctt tagcatctgc tttgttgaag agatacgcta tctcaagatt ttgtcatgtt
     5341 ccatggtttc aagctaaaac tacaagagat gcaagaacta aaccagtttt tgttttgcca
     5401 tctggtgacg aaccattgat tttcaatgtt tcacatcaag caggtttggc tgttttgtta
     5461 gcagttcatg atccaccaaa aggtttagct attggtgttg atgttgtttg tccatctgaa
     5521 agaagagata gagatttgtc ttcattagaa gaagatggtt gggcatcatt cgttgatatc
     5581 catgcagatg tttttggtgc tggtgaagtt tctgcattga aatcaatgaa tccagttcca
     5641 actgctcaag atagagatag agcattgaga tacttctacg ctttgtggtg tttgagagaa
     5701 gcttacgtta agatgacagg tgacgcattg ttagcttctt ggttgaagga tttggaaatg
     5761 cataacttcg caccaccaga agatatgaaa gaagctcaag aagttagatt gagaggtaaa
     5821 aaggttgaag gtgttgatgt tagattggtt ccattgttgg aagaatacat gatctctaca
     5881 gctatcagaa acggtgacaa cggtgaatca attaaattag gtgaatttca aaacttaaac
     5941 atggaagaag tattggaatt cggtgaaaag gctcctaaat cataaagctt ggtaccgcgg
     6001 ctagctaaga tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     6061 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     6121 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     6181 ggttttggga cgctcgaaga tccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6241 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6301 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6361 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6421 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6481 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6541 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6601 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6661 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6721 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6781 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6841 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     6901 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6961 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7021 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7081 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7141 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7201 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7261 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7321 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7381 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7441 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7501 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7561 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7621 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7681 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7741 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7801 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7861 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7921 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7981 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8041 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     8101 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     8161 tccgcgcaca tttccccgaa aagtgccacc tgaacgaagc atctgtgctt cattttgtag
     8221 aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta
     8281 cagaacagaa atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt
     8341 tgtaaaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat
     8401 ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc
     8461 ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat
     8521 tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta
     8581 ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc
     8641 ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga
     8701 taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa
     8761 agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg
     8821 tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat
     8881 gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa
     8941 tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata
     9001 gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg
     9061 tattcgcaat attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc
     9121 gtcttcagag cgcttttggt tttcaaaagc gctctgaagt tcctatactt tctagagaat
     9181 aggaacttcg gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa
     9241 cgcgagctgc gcacatacag ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt
     9301 atatatatat acatgagaag aacggcatag tgcgtgttta tgcttaaatg cgtacttata
     9361 tgcgtctatt tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt atcccattcc
     9421 atgcggggta tcgtatgctt ccttcagcac taccctttag ctgttctata tgctgccact
     9481 cctcaattgg attagtctca tccttcaatg ctatcatttc ctttgatatt ggatcatact
     9541 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     9601 gtc
//
