LOCUS       sequence-232673-        7663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast integrative vector for the exression of the 40nm GEM under the
            INO4 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7663)
  AUTHORS   Delarue M, Brittingham GP, Pfeffer S, Surovtsev IV, Pinglay S, 
            Kennedy KJ, Schaffer M, Gutierrez JI, Sang D, Poterewicz G, Chung 
            JK, Plitzko JM, Groves JT, Jacobs-Wagner C, Engel BD, Holt LJ
  TITLE     mTORC1 Controls Phase Separation and the Biophysical Properties of 
            the Cytoplasm by Tuning Crowding.
  JOURNAL   Cell. 2018 Jul 12;174(2):338-349.e20. doi: 
            10.1016/j.cell.2018.05.042. Epub 2018 Jun 21.
  PUBMED    29937223
REFERENCE   2  (bases 1 to 7663)
  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: 
            "10.1016/j.cell.2018.05.042"; journalName: "Cell"; date: 
            "2018-07-12- 12"; volume: "174"; issue: "2"; pages: "338-349.e20"
COMMENT     Sequence Label: pRS305-Leu2-PINO4-PfV-GS-Sapphire
FEATURES             Location/Qualifiers
     source          1..7663
                     /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     3086..3105
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3086..3104
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4390..5106
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     terminator      5127..5314
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5182..5206)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(5403..5421)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5442..5458)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5442..5458)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5455..5477)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5466..5482
                     /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(5490..5496,5497..5514,5515..5520))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5535..5556
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5673..5690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5844..6432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5924..5943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6603..7394,7395..7463))
                     /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     7226..7245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7464..7568)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7636..7654
                     /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 aattgtaata cgactcacta tagggcgaat tggagctctc
     3121 ttggcttggt ttgtatgagc ttgttacgta tcaaacaaaa tattcacatg tttttctcac
     3181 cttaaagaca gaggcgcgat tgcgtttgcg aaattccaat gccaacaaga taagcttgac
     3241 gaaggagtta agagggcggc ttgaactaaa aagagaaaag caatatctag aatgctctca
     3301 ataaatccaa cccttataaa tagggataag ccatacacca aagaagaatt aatggaaatt
     3361 ctgagattag caataatagc tgagcttgat gcaataaatc tatatgagca gatggcaaga
     3421 tattccgaag atgaaaacgt aagaaagatt ctccttgatg ttgcaaggga ggagaaggct
     3481 cacgttggag aattcatggc actccttcta aatttggatc cagaacaagt aactgagctg
     3541 aagggtggat ttgaggaagt caaggagcta actggaattg aggctcacat aaatgataac
     3601 aagaaggagg aatctaacgt ggaatatttt gagaagttga gaagtgctct tttggatggg
     3661 gtaaataagg gaagatcttt actaaagcac ttgccagtaa cgagaataga aggacaatca
     3721 ttcagggttg acatcattaa atttgaagat ggtgttaggg tagtaaagca agagtacaag
     3781 ccaattccat tattgaagaa gaaattctat gtgggaataa gagaactcaa cgatgggact
     3841 tacgatgtat ctatagcaac taaagctggg gaactcctag ttaaagacga agagtcgcta
     3901 gtaataagag aaattctatc cacagaggga attaagaaaa tgaaactttc ctcctgggac
     3961 aatccagaag aagcacttaa tgatttaatg aacgcactgc aggaagcctc aaatgcgtca
     4021 gctggaccat ttgggctgat aattaaccca aaaagatatg caaagttgct aaaaatttac
     4081 gagaagagtg gaaagatgct agttgaagtg ctaaaagaga tattcagggg aggaatcatt
     4141 gttactctaa acattgatga gaataaggta ataatatttg ccaatacacc agctgttctt
     4201 gacgttgtag ttggccaaga tgtgactcta caagagctag gacctgaagg agacgacgtt
     4261 gcattcttgg ttagtgaagc tataggaata agaataaaga acccagaggc aatagtagtc
     4321 ttagagtggt ctggcagcgg tagtgggtcg ggatcaggct ccggtcgacg gatccccggg
     4381 ttaattaaga tgtctaaagg tgaagaatta ttcactggtg ttgtcccaat tttggttgaa
     4441 ttagatggtg atgttaatgg tcacaaattt tctgtctccg gtgaaggtga aggtgatgct
     4501 acttacggta aattgacctt aaaatttatt tgtactactg gtaaattgcc agttccatgg
     4561 ccaaccttag tcactacttt ttcttatggt gttcaatgtt ttgctagata cccagatcat
     4621 atgaaacaac atgacttttt caagtctgcc atgccagaag gttatgttca agaaagaact
     4681 atttttttca aagatgacgg taactacaag accagagctg aagtcaagtt tgaaggtgat
     4741 accttagtta atagaatcga attaaaaggt attgatttta aagaagatgg taacatttta
     4801 ggtcacaaat tggaatacaa ctttaactct cacaatgttt acatcatggc tgacaaacaa
     4861 aagaatggta tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg ttctgttcaa
     4921 ttagctgacc attatcaaca aaatactcca attggtgatg gtccagtctt gttaccagac
     4981 aaccattact tatccattca atctgcctta tccaaagatc caaacgaaaa gagagaccac
     5041 atggtcttgt tagaatttgt tactgctgct ggtattaccc atggtattga tgaattgtac
     5101 aaataaggcg cgccacttct aaataagcga atttcttatg atttatgatt tttattatta
     5161 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
     5221 gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt
     5281 atgaggtcgc tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa
     5341 tatagatctg tttagcttgc ctcgtccccg ctcgaggggg ggcccggtac cagcttttgt
     5401 tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5461 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5521 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5581 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5641 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5701 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5761 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5821 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5881 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5941 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6001 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6061 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6121 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6181 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6241 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6301 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6361 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6421 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6481 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6541 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6601 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6661 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6721 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6781 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6841 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6901 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6961 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7021 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7081 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7141 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7201 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7261 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7321 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7381 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7441 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7501 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7561 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     7621 acattaacct ataaaaatag gcgtatcacg aggccctttc gtc
//
