LOCUS       sequence-360440-        8258 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative plasmid for expressing ERG20(F96W-N127W) (GAL10 
            promoter) and limonene synthase from Citrus limon (ClLIS1; GAL7 
            promoter). Contains uracil auxotrophic marker (K. lactis URA3)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8258)
  AUTHORS   Cheah LC, Liu L, Stark T, Plan MR, Peng B, Lu Z, Schenk G, Sainsbury
            F, Vickers CE
  TITLE     Metabolic flux enhancement from the translational fusion of terpene 
            synthases is linked to terpene synthase accumulation.
  JOURNAL   Metab Eng. 2023 May;77:143-151. doi: 10.1016/j.ymben.2023.03.012. 
            Epub 2023 Mar 28.
  PUBMED    36990382
REFERENCE   2  (bases 1 to 8258)
  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: "2023-05"; volume: "77:143-151. doi"; pages: " 
            10.1016/j.ymben.2023.03.012. Epub 2023 Mar 28"
COMMENT     Sequence Label: Coexpressed Free ClLIS1 (M)
FEATURES             Location/Qualifiers
     source          1..8258
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(126..567)
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     primer_bind     complement(132..155)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    450..567
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(5521..5538)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5521..5537)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5530..5552)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5746..5765)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5865..5887
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5925..5943)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6011..6115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6116..6184,6185..6976)
                     /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(6334..6353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7147..7735
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7636..7655
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7889..7906
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8023..8044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8059..8064,8065..8082,8083..8089)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8097..8113
                     /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     8102..8124
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8121..8137
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8121..8137
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
       61 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctac tagttatagt
      121 tttttctcct tgacgttaaa gtatagaggt atattaacaa ttttttgttg atacttttat
      181 gacatttgaa taagaagtaa tacaaaccga aaatgttgaa agtattagtt aaagtggtta
      241 tgcagctttt gcatttatat atctgttaat agatcaaaaa tcatcgcttc gctgattaat
      301 taccccagaa ataaggctaa aaaactaatc gcattattat cctatggttg ttaatttgat
      361 tcgttgattt gaaggtttgt ggggccaggt tactgccaat ttttcctctt cataaccata
      421 aaagctagta ttgtagaatc tttattgttc ggagcagtgc ggcgcgaggc acatctgcgt
      481 ttcaggaacg cgaccggtga agaccaggac gcacggagga gagtcttccg tcggagggct
      541 gtcgcccgct cggcggcttc taatccgtac ttcaatatag caatgagcag ttaagcgtat
      601 tactgaaagt tccaaagaga aggttttttt aggctaagat aatggggctc tttacatttc
      661 cacaacatat aagtaagatt agatatggat atgtatatgg tggtattgcc atgtaatatg
      721 attattaaac ttctttgcgt ccatccaaaa aaaaaagtaa gaatttttga aaattcaata
      781 taaggatcca aaatggcttc agaaaaagaa attaggagag agagattctt gaacgttttc
      841 cctaaattag tagaggaatt gaacgcatcg cttttggctt acggtatgcc taaggaagca
      901 tgtgactggt atgcccactc attgaactac aacactccag gcggtaagct aaatagaggt
      961 ttgtccgttg tggacacgta tgctattctc tccaacaaga ccgttgaaca attggggcaa
     1021 gaagaatacg aaaaggttgc cattctaggt tggtgcattg agttgttgca ggcttactgg
     1081 ttggtcgccg atgatatgat ggacaagtcc attaccagaa gaggccaacc atgttggtac
     1141 aaggttcctg aagttgggga aattgccatc tgggacgcat tcatgttaga ggctgctatc
     1201 tacaagcttt tgaaatctca cttcagaaac gaaaaatact acatagatat caccgaattg
     1261 ttccatgagg tcaccttcca aaccgaattg ggccaattga tggacttaat cactgcacct
     1321 gaagacaaag tcgacttgag taagttctcc ctaaagaagc actccttcat agttactttc
     1381 aagactgctt actattcttt ctacttgcct gtcgcattgg ccatgtacgt tgccggtatc
     1441 acggatgaaa aggatttgaa acaagccaga gatgtcttga ttccattggg tgaatacttc
     1501 caaattcaag atgactactt agactgcttc ggtaccccag aacagatcgg taagatcggt
     1561 acagatatcc aagataacaa atgttcttgg gtaatcaaca aggcattaga acttgcttcc
     1621 gcagaacaaa gaaagacttt agacgaaaat tacggtaaga aggactcagt cgcagaagcc
     1681 aaatgcaaaa agattttcaa tgacttgaaa attgaacagc tataccacga atatgaagag
     1741 tctattgcca aggatttgaa ggccaaaatt tctcaggtcg atgagtctcg tggcttcaaa
     1801 gctgatgtct taactgcgtt cttgaacaaa gtttacaaga gaagcaaata aagatctttt
     1861 gccagcttac tatccttctt gaaaatatgc actctatatc ttttagttct taattgcaac
     1921 acatagattt gctgtataac gaattttatg ctatttttta aatttggagt tcagtgataa
     1981 aagtgtcaca gcgaatttcc tcacatgtag ggaccgaatt gtttacaagt tctctgtacc
     2041 accatggaga catcaaaaat tgaaaatcta tggaaagata tggacggtag caacaagaat
     2101 atagcacgag ccgcggagtt catttcgtta cttttgatat cactcacaac tattgcgaag
     2161 cgcttcagtg aaaaaatcat aaggaaaagt tgtaaatatt attggtagta ttcgtttggt
     2221 aaagtagagg gggtaatttt tcccctttat tttgttcata cattcttaaa ttgctttgcc
     2281 tctccttttg gaaagctata cttcggagca ctgttgagcg aaggctcatt agatatattt
     2341 tctgtcattt tccttaaccc aaaaataagg gaaagggtcc aaaaagcgct cggacaactg
     2401 ttgaccgtga tccgaaggac tggctataca gtgttcacaa aatagccaag ctgaaaataa
     2461 tgtgtagcta tgttcagtta gtttggctag caaagatata aaagcaggtc ggaaatattt
     2521 atgggcatta ttatgcagag catcaacatg ataaaaaaaa acagttgaat attccctcaa
     2581 aagagctcaa aatgagaaga tcagctaact atcaaccatc catttgggac cacgactttt
     2641 tacaatcctt gaactctaac tacaccgacg aagcatacaa gagaagagca gaagaattac
     2701 gtggtaaagt aaagatagcc atcaaagatg tcatcgaacc tttggaccaa ttggaattga
     2761 ttgataactt gcaaagattg ggtttagccc atagatttga aaccgaaatc agaaacatct
     2821 tgaacaacat ctataacaac aataaggatt acaactggag aaaggaaaat ttgtacgcta
     2881 cttccttgga attcagattg ttaagacaac acggttaccc agtcagtcaa gaagttttta
     2941 acggtttcaa ggatgaccaa ggtggtttta tttgtgatga cttcaagggt atattgtcct
     3001 tgcatgaagc ttcttactac tcattggaag gtgaaagtat aatggaagaa gcatggcaat
     3061 tcacttccaa acacttgaag gaagttatga taagtaaaaa tatggaagaa gatgttttcg
     3121 tagctgaaca agcaaagaga gccttggaat taccattgca ttggaaagtt cctatgttgg
     3181 aagcaagatg gttcatccat atatacgaaa gaagagaaga taagaaccac ttgttgttgg
     3241 aattggcaaa gatggaattc aatacattac aagccatcta tcaagaagaa ttgaaggaaa
     3301 tctctggttg gtggaaggat accggtttgg gtgaaaagtt gtcattcgct agaaatagat
     3361 tggtcgcatc tttcttatgg tcaatgggta ttgcctttga acctcaattc gcttactgta
     3421 gaagagtttt gacaatctct attgcattga tcaccgttat agatgacatc tatgacgtat
     3481 acggtacttt ggatgaattg gaaatcttta cagacgctgt tgaaagatgg gatatcaact
     3541 atgcattaaa gcatttgcca ggttacatga agatgtgctt tttagccttg tacaacttcg
     3601 tcaacgaatt cgcttactac gttttgaagc aacaagattt cgacttattg ttatctatta
     3661 aaaacgcttg gttgggtttg atacaagcct atttggtaga ggctaagtgg taccattcta
     3721 agtacacacc taagttggaa gaatacttgg aaaacggttt agtctcaatc actggtccat
     3781 tgatcatcac aatctcctat ttgagtggta ctaaccctat cattaaaaag gaattggaat
     3841 tcttggaatc aaacccagat atcgttcact ggtcttcaaa aattttcaga ttgcaagatg
     3901 acttaggtac ttccagtgac gaaattcaaa gaggtgacgt acctaaatct atacaatgct
     3961 acatgcatga aacaggtgca tcagaagaag ttgccagaca acacattaag gatatgatga
     4021 gacaaatgtg gaaaaaggtt aatgcttaca ccgcagataa agactcccca ttgactggta
     4081 ctacaaccga attcttgttg aacttagtta gaatgtctca tttcatgtat ttgcatggtg
     4141 acggtcacgg tgtacaaaat caagaaacca ttgatgtcgg ttttactttg ttattccaac
     4201 ctattccatt agaagataaa cacatggcat tcaccgcatc accaggtaca aagggttaat
     4261 agctcgagat agattaattt aaacagtata tgtacagttt tatatatata tatatatata
     4321 tacatatata aagaaacctg tgcgtttttt gtattttcaa atacatttag ttttggcgcc
     4381 acttctataa aaggtctcat gatattgtta ctctagagat cccaatacaa cagatcacgt
     4441 gatctttttg taagatgaag ttgaagtagt gttgcaccgt gccaatgcag gtggctatta
     4501 gattaaatat gtgatttgtt ctattaagtt tcctgtataa ttaatgggga gcgctgattc
     4561 tcttttggta cgcttcccat ccagcatttc tgtatctttc accttcaacc ttaggatctc
     4621 tacccttggc gaaaagtcct ctgccaacaa tgatgatatc tgatccacca cttacaactt
     4681 cgtcgacggt tctgtactgc tgacccaatg catcgccttt gtcgtctaaa cctacacctg
     4741 gggtcatgat tagccaatca aacccttctt ctcttcctcc catatcgttc tgagcaatga
     4801 acccaataac gaaatcttta tcactctttg caatatcaac ggtaccctta gtatattcac
     4861 cgtgtgctag agaacccttg gaagacaatt cagcaagcat caataatccc cttggttctt
     4921 tggtgacctc ttgcgcacct tgtttcaagc cagcaacaat accagcacca gtaaccccgt
     4981 gggcgttggt gatatcagac cattctgcga tacggtaaac gcccgatgta tattgtaatt
     5041 tgactgtgtt accgatatcg gcgaattttc tgtcctcaaa tatcaagaac ttgtatttct
     5101 ctgccaatgc tttcaatgga acgacagtac cctcataact gaaatcatcc aagatatcaa
     5161 cgtgtgtttt caaaaggcaa atgtatggac ccaacgtttc aacaagtttc aatagctcat
     5221 cagtcgaacg aacgtcaaga gaagcacaca aattggtctt cttttcatcc attaaacgta
     5281 aaagtttcga tgcaaccgga cttgcatgag tctcagctct actggtatat gattttgtgg
     5341 acatgtttta tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt aagaatactg
     5401 ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta agtctgtgct
     5461 ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt aaatgaattc
     5521 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5581 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5641 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     5701 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     5761 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5821 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5881 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     5941 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     6001 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6061 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6241 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6301 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6361 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6421 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6481 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6541 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6601 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6661 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6721 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6781 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6841 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6901 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6961 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7021 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7141 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7201 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7261 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     7321 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     7381 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7441 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7501 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     7561 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     7621 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     7681 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     7741 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     7801 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     7861 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     7921 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     7981 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     8041 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     8101 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcttgcatg
     8161 catttaaatc attacgaccg agattcccgg gtaataactg atataattaa attgaagctc
     8221 taatttgtga gtttagtata catgcattta cttataat
//
