LOCUS       sequence-136659-        8921 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Inducible expression of EGFP in yeast. Has dual-mode promoter with 
            activation and repression capability for tuning gene expression in 
            yeast. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8921)
  AUTHORS   Mazumder M, McMillen DR
  TITLE     Design and characterization of a dual-mode promoter with activation 
            and repression capability for tuning gene expression in yeast.
  JOURNAL   Nucleic Acids Res. 2014 Aug;42(14):9514-22. doi: 10.1093/nar/gku651.
            Epub 2014 Jul 23.
  PUBMED    25056312
REFERENCE   2  (bases 1 to 8921)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1093/nar/gku651"; journalName: "Nucleic Acids Res"; date: 
            "2014-08"; volume: "42"; issue: "14"; pages: "9514-22"
COMMENT     Sequence Label: pLAREG
FEATURES             Location/Qualifiers
     source          1..8921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(663..1757)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVAGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANLLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1770..2174)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2474..2929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2823..2842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3011..3200)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     terminator      3318..3507
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     CDS             complement(3538..4254)
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     protein_bind    4365..4381
                     /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)."
     promoter        5090..5531
                     /label=TPGI promoter
                     /note="S. cerevisiae GAL1 promoter modified to contain two 
                     copies of the tet operator (Ellis et al., 2009)"
     protein_bind    5090..5207
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     protein_bind    5415..5433
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5436..5454
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     5502..5525
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     terminator      6670..6859
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6931..6948)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7102..7690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7182..7201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7861..8652,8653..8721))
                     /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     8484..8503
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8722..8826)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8894..8912
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
      661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
      721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
      781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
      841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
      901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
      961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
     1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
     1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
     1141 acagtttttc tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata
     1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
     1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
     1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
     1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
     1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
     1501 ctaccggtac cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg
     1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
     1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
     1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
     1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
     1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
     1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
     1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
     1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
     2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
     2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
     2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
     2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
     2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
     2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
     2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
     2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3001 accaggtgat cttcgagcgt cccaaaacct tctcaagcaa ggttttcagt ataatgttac
     3061 atgcgtacac gcgtctgtac agaaaaaaaa gaaaaatttg aaatataaat aacgttctta
     3121 atactaacat aactataaaa aaataaatag ggacctagac ttcaggttgt ctaactcctt
     3181 ccttttcggt tagagcggat cttagctagc cgcggtacca agcttactcg agttaagacc
     3241 cactttcaca tttaagttgt ttttctaatc cgcatatgat caattcaagg ccgaataaga
     3301 aggctggctc tgcacctatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     3361 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     3421 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     3481 cttgagaagg ttttgggacg ctcgaagatc cagctggcgt aatagcgaag aggcccgtta
     3541 tttgtacaat tcatccatac catgggtaat accagcagca gtaacaaatt ctaacaagac
     3601 catgtggtct ctcttttcgt ttggatcttt ggataaggca gattgagtgg ataagtaatg
     3661 gttgtctggt aacaagactg gaccatcacc aattggagta ttttgttgat aatggtcagc
     3721 taattgaaca gaaccatctt caatgttgtg tctaattttg aagttaactt tgataccatt
     3781 cttttgtttg tcagccatga tgtaaacatt gtgagagtta tagttgtatt ccaatttgtg
     3841 acctaaaatg ttaccatctt ctttaaaatc aatacctttt aattcgattc tattaactaa
     3901 ggtatcacct tcaaacttga cttcagctct ggtcttgtag ttaccgtcat ctttgaaaaa
     3961 aatagttctt tcttgaacat aaccttctgg catggcagac ttgaaaaagt catgttgttt
     4021 catatgatct gggtatctcg caaaacattg aacaccataa ccgaaagtag tgactaaggt
     4081 tggccatgga actggcaatt taccagtagt acaaataaat tttaaggtca atttaccgta
     4141 agtagcatca ccttcacctt caccggagac agaaaatttg tgaccattaa catcaccatc
     4201 taattcaacc aaaattggga caacaccagt gaataattct tcacctttag acattattaa
     4261 tttagtgtgt gtatttgtgt ttgcgtgtct atagaagtat agtaatttat gctacaaagg
     4321 acctaatgta taaggaaaga atatttagag aaaagtgtgt gaaattgtta tccgctcaca
     4381 attccacaca aagaaaacaa gagttttata tacatacaga gcacatgcct cgaggtcgac
     4441 cccgggaaaa agaacaccct gtaccagaca aaaagaacac cctgtaccag acgtcgacaa
     4501 aaagaacacc ctgtaccaga caaaaagaac accctgtacc agacaaaaag aacaccctgt
     4561 accagacgga tctactagtg cggccgcatc ttgactgatt tttccatgga gggcacagtt
     4621 aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt
     4681 gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa
     4741 tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag
     4801 caggcggcag aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca
     4861 tgcaagggct ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     4921 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     4981 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt
     5041 caacagtata gaaccgtgga tgatgtggtc tctacaggaa ttcgaagtac ggattagaag
     5101 ccgccgagcg ggtgacagcc ctccgaagga agactctcct ccgtgcgtcc tcgtcttcac
     5161 cggtcgcgtt cctgaaacgc agatgtgcct cgcgccgcac tgctccgaac aataaagatt
     5221 ctacaatact agcttttatg gttatgaaga ggaaaaattg gcagtaacct ggccccacaa
     5281 accttcaaat gaacgaatca aattaacaac cataggatga taatgcgatt agttttttag
     5341 ccttatttct ggggtactgc agcagcgaag cgatgatttt tgatctatta acagatatat
     5401 aaatgcaaaa actgtcccta tcagtgatag agatctccct atcagtgata gagatattac
     5461 ttcttattca aatgtaataa aagtatcaac aaaaaattgt taatatacct ctatacttta
     5521 acgtcaagga gaaaaaaccc cggatcctat taaaatgaaa ccagtaacat tgtatgatgt
     5581 cgcagagtat gccggtgtct cttatcagac tgtttccaga gtggtgaacc aggccagcca
     5641 tgtttctgcc aaaaccaggg aaaaagtgga agcagccatg gcagagctga attacattcc
     5701 caacagagtg gcacaacaac tggcaggcaa acagagcttg ctgattggag ttgccacctc
     5761 cagtctggcc ctgcatgcac catctcaaat tgtggcagcc attaaatcta gagctgatca
     5821 actgggagcc tctgtggtgg tgtcaatggt agaaagaagt ggagttgaag cctgtaaagc
     5881 tgctgtgcac aatcttctgg cacaaagagt cagtgggctg atcattaact atccactgga
     5941 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccagcac tctttcttga
     6001 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagatg gtacaagact
     6061 gggtgtggag catctggttg cattgggaca ccagcaaatt gcactgcttg cgggcccact
     6121 cagttctgtc tcagcaaggc tgagactggc cggctggcat aaatatctca ctaggaatca
     6181 aattcagcca atagctgaaa gagaagggga ctggagtgcc atgtctgggt ttcaacaaac
     6241 catgcaaatg ctgaatgagg gcattgttcc cactgcaatg ctggttgcca atgatcagat
     6301 ggcactgggt gcaatgagag ccattactga gtctgggctg agagttggtg cagatatctc
     6361 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     6421 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     6481 gggccaggcg gtgaagggca atcagctgtt gccagtctca ctggtgaaga gaaaaaccac
     6541 cctggcaccc aatacacaaa ctgcctctcc ccgggcattg gctgattcac tcatgcagct
     6601 agcaagacag gtttccagac tggaaagtgg gcagtgactc gagtaagctt ggtaccgcgg
     6661 ctagctaaga tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     6721 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     6781 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     6841 ggttttggga cgctcgaaga tccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6901 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6961 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     7021 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     7081 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     7141 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     7201 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     7261 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     7321 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     7381 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     7441 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7501 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     7561 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7621 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7681 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7741 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7801 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7861 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7921 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7981 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     8041 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     8101 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     8161 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     8221 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     8281 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     8341 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     8401 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     8461 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     8521 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     8581 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     8641 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8701 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     8761 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     8821 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     8881 attaacctat aaaaataggc gtatcacgag gccctttcgt c
//
