LOCUS       sequence-405744-        7836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing the TEV protease, codon-optimized for 
            Saccharomyces cerevisiae..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7836)
  AUTHORS   Geistodt-Kiener A, Totozafy JC, Le Goff G, Vergne J, Sakai K, 
            Ouazzani J, Mouille G, Viaud M, O'Connell RJ, Dallery JF
  TITLE     Yeast-based heterologous production of the colletochlorin family of 
            fungal secondary metabolites.
  JOURNAL   Metab Eng. 2023 Oct 18:S1096-7176(23)00142-8. doi: 
            10.1016/j.ymben.2023.10.002.
  PUBMED    37863177
REFERENCE   2  (bases 1 to 7836)
  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. 
            2023 Oct 18:S1096-7176(23)00142-8. doi: 
            10.1016/j.ymben.2023.10.002."
COMMENT     Sequence Label: pHYX173
FEATURES             Location/Qualifiers
     source          1..7836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..57)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(100..111)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     389..408
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             complement(748..768)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1312..1328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1312..1328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1340..1357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1511..2099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1591..1610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2347..3162)
                     /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="MSHIQRETSCSRPHLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3070..3089
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3154..3173
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        complement(3456..3560)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3587..4927
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4550..4597)
                     /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     4968..4986
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5024..5046)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5146..5165
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5189..5404
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             5405..6208
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     CDS             complement(6964..6984)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7024..7080)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(7099..7119)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(7123..7830)
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
ORIGIN
        1 agggccaggg ttttcttcaa cgtcgccagc ctgtttgagg agagagaagt tggtggcgcc
       61 agagccgttt aaacattgag cgagctcctt cttcagggcc tgcagctccc acttcagctg
      121 cgccagctcc ttcttgttgg cctgcagctc cttcttcagg gccccgggtc ctccgccaga
      181 tccgccgcct cctctctcga tgttgtggcg aatcttgaag ttggccttga tgccgttctt
      241 ctgcttgtcg gcggtgatgt agacgttgtg ggagttgtag ttgtactcga gcttgtggcc
      301 gaggatgttg ccatcttcct tgaagtcgat gcccttcagc tcgatgcggt tgacgagggt
      361 gtctccctcg aacttgacct cagcgcgggt cttgtagttg ccgtcgtcct tgaagaaaat
      421 ggtgcgctct tggacgtagc cctcaggcat ggcggacttg aagaagtcgt gctgcttcat
      481 gtggtcaggg tagcgggcga agcactggag gccgtagccg agggtggtga cgagggtagg
      541 ccaagggaca gggagcttgc cggtggtgca gatgagcttg agggtgagct tgccgtaggt
      601 ggcgtcgccc tcgccctcgc cgctgacgga gaacttgtgg ccgttgacgt cgccgtcaag
      661 ctcgacgagg atagggacga cgccggtgaa gagttcctcg cccttgctga ccatcccggg
      721 gccggagccg ccggcggtac ccaattcgac ctttctcttc ttttttggag gctcgggaat
      781 taattccgct ttatccatgt tgttatttta ttatggaata attagttgcg tgagtttggt
      841 tttttttttt ttatttttga gttaaacaaa tataattatt gtttaagctg aaataaagat
      901 atatatgtat atctgggaaa ggctgttggt tatctgtctt acttctgtat atttaagaag
      961 taagtagaaa tactgattac acatggaaga gaagttacac acattacaaa gacttctctg
     1021 cccttatata cctttttttt gtggtcttct atactctgcc caatgttgct atccccagcc
     1081 aagccttact taatacacaa acgtaccatt gtccaaccag aagcgtctaa gctttacaat
     1141 cctgtttggg ccctttatcc gcctatccat aatgttaagc agcctaaaac ttgctagtct
     1201 agccggcatc tttcccttgt ataacttaaa atacccggag cttcagtgat atgtagaaca
     1261 cagccgcgcc ggatgaaagg aagctcggtt ttttcctatg cccgggccat ggtcatagct
     1321 gtttcctgtg tgacgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     1381 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     1441 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     1501 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     1561 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1621 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1681 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1741 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1801 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1861 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1921 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     1981 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     2041 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     2101 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     2161 ggattttggt catgaccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2221 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2281 ataccgctcg ccgcgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa
     2341 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     2401 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     2461 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     2521 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     2581 gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac
     2641 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2701 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     2761 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     2821 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     2881 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     2941 ccagtttaaa cggaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     3001 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     3061 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     3121 atgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     3181 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     3241 acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt
     3301 gaaggatcag atcacgcatc ttcccgaccc actcgtgcac ccaactgatc ttcagcatct
     3361 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3421 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3481 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3541 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaacg aagcatctgt
     3601 gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
     3661 gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat
     3721 ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
     3781 aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa
     3841 agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac
     3901 aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa
     3961 ctttttgcac tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct
     4021 tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt
     4081 gcattttttc aagataaagg catccccgat tatattctat accgatgtgg attgcgcata
     4141 ctttgtgaac agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg
     4201 tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt
     4261 tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag
     4321 ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat
     4381 gggtaggtta tatagggata tagcacagag atatatagca aagagatact tttgagcaat
     4441 gtttgtggaa gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg
     4501 ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat
     4561 actttctaga gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc
     4621 ttccgaaaat gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc
     4681 tgcgtgttgc ctgtatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa
     4741 tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata
     4801 ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta
     4861 tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata
     4921 ttggatcata ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4981 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     5041 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     5101 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     5161 ttgtactgag agtgcaccat accacctttt caattcatca tttttttttt attctttttt
     5221 ttgatttcgg tttccttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg
     5281 aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg
     5341 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata
     5401 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc
     5461 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg
     5521 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa
     5581 aacacatgtg gatattttga ctgatttttc gatggagggc acagttaagc cgctaaaggc
     5641 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa
     5701 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac
     5761 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga
     5821 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct
     5881 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt
     5941 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat
     6001 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac
     6061 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc
     6121 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata
     6181 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact
     6241 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa
     6301 taccgcacag atgcggcccg ggcgctatat gtacgttgtt agtgccgtta ataataaatt
     6361 catcctctat catagaaaca gggcatgaga aacgaagaaa aattgatatt gtaacaatat
     6421 catgagacct tttatagaag tggcgccaaa actaaatgta tttgaaaata caaaaaacgc
     6481 acaggtttct ttatatatgt atatatatat atatatatat ataaaactgt agatatactg
     6541 tttaaattaa tctatctact tgtacagctc gtccatgccg agggtgatgc cagcggcggt
     6601 gacgaactcg agcaggacca tgtggtcgcg cttctcgtta gggtccttgg agagggcgga
     6661 ctggtagctg aggtagtggt tgtcagggag gaggacaggg ccgtcgccga taggggtgtt
     6721 ctgctggtag tggtcggcga gctggacgcc gccgtcctcg atgttgtggc gaatcttgaa
     6781 gttggccttg atgccgttct tctgcttgtc agagccgcct ccgcctgagc ctcctccgcc
     6841 cccgggttgg gccagcttct tctccagggc ctggttcttc cactccagct gggccagctt
     6901 cttctccagg gcctgcagct tcttctccag ctgctcgccg gagccgccgg cggtgcccag
     6961 ctccaccttg cgcttcttct tggggggctc gcggatcagc tcggccttgt ccatatttaa
     7021 atgagggcca gggttttctt caacgtcgcc agcctgtttg aggagagaga agttggtggc
     7081 gccagagcct cccatcgcac cttgaaaata aagattttcg atattcatga gttgagtcgc
     7141 ttccttaact ggctgaaaag gctcttcagg tttcaccatg aaaactttat ggccccccca
     7201 caatactgag tcagcattta atcgccaacc actaacccac tgctgcgcct cctgatttgt
     7261 caacaattcc atgaagtttt tcggcacgct tgtgaaataa ttgtttgtgt tggtgaaatt
     7321 cgatgctgag tgtataccaa caatgaaccc atctctagtt gatactaatg gactgccaca
     7381 ctgcccatcc ttggtttgaa tccaatgctt ccagaatatg ccatcagatg aagggaatgt
     7441 gcaactagtg tctgacacca tgctagacat gctcttagtt tggaagttgg ttgtcacaag
     7501 acatatgcgc tcttcccttt gtggctctct aaatttcagc ttttgaggaa atggtgggaa
     7561 atccttaggc atgcgaataa ttatcatgtc cctcccatca atgaggtgtt gttgcaaagt
     7621 cgtggtgttc ttgaccttga atacaccatg tagtgattgg accaacagtg ttccattatt
     7681 tcttctaaac aagtgcttgt ttgtaatgat gaagggacca aatccaatac catacaacga
     7741 tgttgtgtgc ccatcagatt cattcgtcaa atgacaaatg gtgctcgata tcgggttgta
     7801 atcacgcggc cccttaaaca agctttctcc catatc
//
