LOCUS       sequence-440009-        6617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Methionine-repressible gene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6617)
  AUTHORS   Oppenheimer KG, Hager NA, McAtee CK, Filiztekin E, Shang C, Warnick 
            JA, Bruchez MP, Brodsky JL, Prosser DC, Kwiatkowski AV, O'Donnell AF
  TITLE     Optimization of the fluorogen-activating protein tag for 
            quantitative protein trafficking and colocalization studies in S. 
            cerevisiae.
  JOURNAL   Mol Biol Cell. 2024 Jul 1;35(7):mr5. doi: 10.1091/mbc.E24-04-0174. 
            Epub 2024 May 29.
  PUBMED    38809589
REFERENCE   2  (bases 1 to 6617)
  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: "Mol Biol 
            Cell."; date: "2024-07-1"; volume: "35(7):mr5. doi"; pages: " 
            10.1091/mbc.E24-04-0174. Epub 2024 May 29"
COMMENT     Sequence Label: pRS415-MET25pr
FEATURES             Location/Qualifiers
     source          1..6617
                     /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(664..1758)
                     /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(1759..2166)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2467..2922)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2604..2625)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2816..2835
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3048..3070
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3062..3079
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3063..3079
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3089..3108
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3089..3107
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(3126..3373)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     3339..3357
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     3375..3391
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3376..3392
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3426..3442)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3426..3442)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(3443..3792)
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     complement(3843..3863)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3843..3861)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3882..3898)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3882..3898)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3895..3917)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3906..3922
                     /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        complement(join(3930..3936,3937..3954,3955..3960))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3975..3996
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4113..4130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4284..4872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4364..4383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5043..5834,5835..5903))
                     /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     5666..5685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5904..6008)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6045..6548
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6590..6608
                     /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 ctacgtcgtt aaggccgttt ctgacagagt aaaattcttg agggaacttt
      241 caccattatg ggaaatggtt caagaaggta ttgacttaaa ctccatcaaa tggtcaggtc
      301 attgagtgtt ttttatttgt tgtatttttt tttttttaga gaaaatcctc caatatataa
      361 attaggaatc atagtttcat gattttctgt tacacctaac tttttgtgtg gtgccctcct
      421 ccttgtcaat attaatgtta aagtgcaatt ctttttcctt atcacgttga gccattagta
      481 tcaatttgct tacctgtatt cctttacatc ctcctttttc tccttcttga taaatgtatg
      541 tagattgcgt atatagtttc gtctacccta tgaacatatt ccattttgta atttcgtgtc
      601 gtttctatta tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc
      661 tttttaagca aggattttct taacttcttc ggcgacagca tcaccgactt cggtggtact
      721 gttggaacca cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc
      781 ttcaatggcc ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga
      841 caagatagtg gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca
      901 tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg
      961 caacaaaccc aaggaacctg ggataacgga ggcttcatcg gagatgatat caccaaacat
     1021 gttgctggtg attataatac catttaggtg ggttgggttc ttaactagga tcatggcggc
     1081 agaatcaatc aattgatgtt gaaccttcaa tgtagggaat tcgttcttga tggtttcctc
     1141 cacagttttt ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat
     1201 aggcaatggt ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac
     1261 ttctggaacg gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct
     1321 cttaccaaag taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc
     1381 aaattgtggc ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct
     1441 taagttggcg tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac
     1501 actaccggta ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt
     1561 ggaggcttcc agcgcctcat ctggaagtgg aacacctgta gcatcgatag cagcaccacc
     1621 aattaaatga ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag
     1681 aaccttaatg gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat
     1741 cttcttaggg gcagacatta gaatggtata tccttgaaat atatatatat atattgctga
     1801 aatgtaaaag gtaagaaaag ttagaaagta agacgattgc taaccaccta ttggaaaaaa
     1861 caataggtcc ttaaataata ttgtcaactt caagtattgt gatgcaagca tttagtcatg
     1921 aacgcttctc tattctatat gaaaagccgg ttccggcgct ctcacctttc ctttttctcc
     1981 caatttttca gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa caaaaaattt
     2041 ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt atgttgagga
     2101 aaaaaataat ggttgctaag agattcgaac tcttgcatct tacgatacct gagtattccc
     2161 acagttaact gcggtcaaga tatttcttga atcaggcgcc ttagaccgct cggccaaaca
     2221 accaattact tgttgagaaa tagagtataa ttatcctata aatataacgt ttttgaacac
     2281 acatgaacaa ggaagtacag gacaattgat tttgaagaga atgtggattt tgatgtaatt
     2341 gttgggattc catttttaat aaggcaataa tattaggtat gtagatatac tagaagttct
     2401 cctcgaccgg tcgatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     2461 atcaggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2521 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2581 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2641 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2701 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2761 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2821 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2881 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct
     2941 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3001 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     3061 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     3121 cggccgcaaa ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt tttcagtata
     3181 atgttacatg cgtacacgcg tctgtacaga aaaaaaagaa aaatttgaaa tataaataac
     3241 gttcttaata ctaacataac tataaaaaaa taaataggga cctagacttc aggttgtcta
     3301 actccttcct tttcggttag agcggatgtg gggggagggc gtgaatgtaa gcgtgacata
     3361 actaattaca tgactcgagg tcgacggtat cgataagctt gatatcgaat tcctgcagcc
     3421 cgggggatcc actagttcta gagtatggat gggggtaata gaattgtatc tatgtatctg
     3481 acgaccctgt attacacgaa ggaaatagta gatgaaaaga caagagagca agaaaaagga
     3541 aaaacttcct ttctaacaga cgctttactt aaccttatat atatcttatt tttttcatcg
     3601 agcgtgttca attggacaag gtgccacctt ttcgacactt cggttattat gttacacagt
     3661 tttcatgagg atggcgcctt gactaactta attagtcatt tgccaaccac cacagttccc
     3721 caatatcgac agcttcacgt gccatttgcc attttgcgat catgtgacct caagagaaaa
     3781 agcaaaaaat aatgagagct aagggattcg aacccttgca tccggagctc cagcttttgt
     3841 tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     3901 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3961 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4021 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4081 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4321 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4381 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4441 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4501 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4561 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4621 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4681 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4741 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4801 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4861 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4921 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4981 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5041 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5101 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5161 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5221 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5281 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5341 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5401 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5461 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5521 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5581 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5641 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5701 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5761 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5821 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5881 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5941 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6001 gttccgcgca catttccccg aaaagtgcca cctgggtcct tttcatcacg tgctataaaa
     6061 ataattataa tttaaatttt ttaatataaa tatataaatt aaaaatagaa agtaaaaaaa
     6121 gaaattaaag aaaaaatagt ttttgttttc cgaagatgta aaagactcta gggggatcgc
     6181 caacaaatac taccttttat cttgctcttc ctgctctcag gtattaatgc cgaattgttt
     6241 catcttgtct gtgtagaaga ccacacacga aaatcctgtg attttacatt ttacttatcg
     6301 ttaatcgaat gtatatctat ttaatctgct tttcttgtct aataaatata tatgtaaagt
     6361 acgctttttg ttgaaatttt ttaaaccttt gtttattttt ttttcttcat tccgtaactc
     6421 ttctaccttc tttatttact ttctaaaatc caaatacaaa acataaaaat aaataaacac
     6481 agagtaaatt cccaaattat tccatcatta aaagatacga ggcgcgtgta agttacaggc
     6541 aagcgatccg tcctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     6601 cacgaggccc tttcgtc
//
