LOCUS       sequence-472631-        8977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression in FL yeast VPS34 D731N mutant in yeast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8977)
  AUTHORS   Rostislavleva K, Soler N, Ohashi Y, Zhang L, Pardon E, Burke JE, 
            Masson GR, Johnson C, Steyaert J, Ktistakis NT, Williams RL
  TITLE     Structure and flexibility of the endosomal Vps34 complex reveals the
            basis of its function on membranes.
  JOURNAL   Science. 2015 Oct 9;350(6257):aac7365. doi: 10.1126/science.aac7365.
  PUBMED    26450213
REFERENCE   2  (bases 1 to 8977)
  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: "Science."; 
            date: "2015-10-9"; volume: "350(6257):aac7365. doi"; pages: " 
            10.1126/science.aac7365"
COMMENT     Sequence Label: pYO290
FEATURES             Location/Qualifiers
     source          1..8977
                     /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"
     promoter        191..472
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             473..1147
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1250..1705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1387..1408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1599..1618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1831..1853
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1845..1862
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1846..1862
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1872..1891
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1872..1890
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4969..4992)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     protein_bind    5173..5290
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     complement(5375..5395)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5375..5393)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5414..5430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5414..5430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5427..5449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5438..5454
                     /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(5462..5468,5469..5486,5487..5492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5507..5528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5645..5662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5816..6404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5896..5915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6575..7366,7367..7435))
                     /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     7198..7217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7436..7540)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7567..8909
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(8530..8577)
                     /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     8950..8968
                     /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 accatagatc aacgacatta ctatatatat aatataggaa gcatttaata gaacagcatc
      241 gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag atattcttta
      301 ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct ttttttgccg
      361 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg
      421 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt
      481 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga
      541 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa
      601 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata
      661 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga
      721 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga
      781 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc
      841 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt
      901 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg
      961 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc
     1021 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg
     1081 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa
     1141 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gccgatctat
     1201 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     1261 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     1321 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     1381 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     1441 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     1501 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     1561 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     1621 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     1681 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     1741 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     1801 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     1861 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagc
     1921 ggtccggcgc gccggccgcc agatcgatct tcgaggggaa ttctcttagg attcgattca
     1981 cattcatctt tttttagcta ctaataggat aaattatagg aatttataac aaattgaaat
     2041 atggcaggca gcaaaattaa aattgtctta gttttttagt gtatagaagt gaatagctat
     2101 ataaagtatg tgtaaagttg gtaacggaac gaaaaataga aaaggatatt acatgggaaa
     2161 acatgttgtt tacggagaaa tgaaaagtat attgtatttt gtacgagcta aaagtacagt
     2221 gggaacaaag tcgacttagg tccgccagta ttgtgccaga ttatgtaaat gatcaatcac
     2281 gataggcagc aaagcattta cactatcatt gattagattc tgaaaatgca ctgtggcatc
     2341 ttcttcggac atattcaaat taaatctctc tcttacacgt aatatagcac cattgggatc
     2401 tattctgata tcaggtatgt tcgacgtctt cattaattca aacaggttta agattaagcc
     2461 tgcattcctt cttaaaatcg aatatgccac aaaacagtag ctgcgaaatt tatcataatt
     2521 tgatgattct gcacccccaa acgcctctat aatttgaggg ggcaatttca ttaatggcgg
     2581 aaaaggtttg gggtcctgac ccaagatata accaaagtct gcgtgaaaaa agtgcccatc
     2641 tggtgtgact agtaagttgt ctaaatgcct attgccgaca cctaagatgt atgtaataac
     2701 acaatagcca gcacaagatt tgacaaaatt atctaaaacc cagccctgta cgcctaatgt
     2761 cgcgttctca tcaggatagt ggagtttaag gtagccaaga ataccgtgat atttgcttag
     2821 tatgctagct aatgtgtcat tagggataaa ttcaatggca ccttcttgtg gtcctgttgc
     2881 caaaattttg tatggtgtca atttcaagtc cacgttttcg ttttttaata actcattcat
     2941 taaacttatg atctgtacta ccagttgatc ttgtctcaaa tcatcgccaa ccttaaacat
     3001 taagtgatat ggttggttta aggtcgtttt gaatgttatt tttagtggcg ataaggagct
     3061 tttaaatacc ttcgaagttt caggacaaac gtcgcatatc aacacatcag ggtctagtgg
     3121 taaagcaatt ggtcgtactt tgacaagagg cctgactttc gtctccaata aatgcactag
     3181 taattccatc tttttggctg tagtatcttt cagcctctta attgtttcac aacattccct
     3241 gagcacattt attagcctga cttgatcgtt cagtatattc cgagattttt tatccagtct
     3301 actccaaaag gaacttaaaa tctggtctaa atatggcttg tcctcagact cggactttaa
     3361 ataccaatag aagaaacttc ctaaccttgg gttgaccagt gctctcctaa tcaaaaactc
     3421 agccaaggga gaaatgacga ttggtagcga ttctgtcccg gaagtttccg attcactcga
     3481 gatggacttt aataattttt ggttggcatg cgatgtagat agtaacatag aatcacccga
     3541 aagcttttgc gaagatacgg catccacaat agtgaattca ctgttagatt tgtcggaaaa
     3601 agtggacagg ttctcaaaac agacagcttc taccaactgt aataagtaca attccagctc
     3661 cttgtctgat gccttcttca gcctatttac ggcgtacgat ctcactgaaa ggtttttaaa
     3721 agttgagcct aatagttcta atgcgtcgtc tatatcaatt tctgcccatg agtccattag
     3781 ttctaaaacc tctactcttt ccgattcttc tctcaaattt gtactctgta ataactttgt
     3841 aagagctttt ttattgttca tcaaataata cctatatttc catattgaac ccttttcatg
     3901 tgctgttaat ttagtgccag gggggtagtt gatgatttta ttcaagtagt ccctcttttt
     3961 aatgtccggc ttcacttgct tgtccaagtt tgcattttta gatgctcttt ccaatctcct
     4021 atacttttcc tctattggat cattgtttgg ttggtcagga tcgtaaaact tcaatgtgga
     4081 gtgatatttg tcccccaaag aaattttgat ttgagggtca ttcctattcg gttctcgtaa
     4141 gcctgtgctt agtccaggat tgttcttcaa agttggaata ttcatttgag tattcataat
     4201 ttccctttcg atgaacacaa caggaagctc taacattgga aattctaagt ttaagacaaa
     4261 ggtgccctga ggccaagtcc tattttctcg ttgtttcctt aatttgctta tagtgatttc
     4321 gtcaagccaa gggagtctcg tgaattcacc ctgaaaatat ttattcaaat tttcttgatc
     4381 tttgttatca gtgaccactt cacagtggtc aatcacatca taacgaaatt ttaaggattc
     4441 aaaccctctt tttaaagtac agtcttttaa gttaaagatg ctcgtttcta gattgaaaaa
     4501 gggaatttgc ttggaaccat tgtattccca taaaataatg cgtaaatgac tgctaaaggt
     4561 aagctgtttg atacgtatgg gcaaagtcaa ccaataatcc caggttctag agtttctgaa
     4621 aggaatatac ggcgtgtata tcggaagagt taaatttctg tttctctctt tatcaaatac
     4681 ctgtaaggac acaattagat cactagaagg gaatacattt gaccctatca gcattaattc
     4741 agggttcagg attttttgag atggcttcaa cagtggctta tgtccttcca atgacttgat
     4801 tttcactttc aggggaacat ccagatcttg tgagacacag aatgtgatgt tgttcagagc
     4861 catggtttat ttatgtgtgt ttattcgaaa ctaagttctt ggtgttttaa aactaaaaaa
     4921 aagactaact ataaaagtag aattcaagaa gtttaagaaa tagatttaca gaattacaat
     4981 caatacctac cgtctttata tacttattag tcaagtaggg gaataatttc agggaactgg
     5041 tttaaacctt ttttttcagt cgagatatct ggttgttaat ttgattcgtt catttgaggt
     5101 ttgtggggcc aggttactgc caattttcct cttcataacc ataaaagcta gtattgtaga
     5161 atctttattg ttcggagcag tgcggcgcga ggcacatctg cgtttcagga acgcgaccgg
     5221 tgaagacgag gacgcacgga ggagagtctt ccttcggagg gctgtcaccc gctcggcggc
     5281 ttctaatccg tacgcatgct ttttttggat ctttgcaaag atccgcggcc ggcgcgccgc
     5341 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     5401 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5461 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5521 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5581 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5641 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5701 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5761 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5821 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5881 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5941 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6001 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6061 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6121 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6181 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6241 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6301 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6361 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6421 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6481 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6541 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6601 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6661 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6721 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6781 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6841 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6901 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6961 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7021 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7081 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7141 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7201 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7261 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7321 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7381 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7441 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7501 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7561 cacctgaacg aagcatctgt gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta
     7621 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg
     7681 ctattttacc aacgaagaat ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag
     7741 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga
     7801 gagcgctatt ttaccaacaa agaatctata cttctttttt gttctacaaa aatgcatccc
     7861 gagagcgcta tttttctaac aaagcatctt agattacttt ttttctcctt tgtgcgctct
     7921 ataatgcagt ctcttgataa ctttttgcac tgtaggtccg ttaaggttag aagaaggcta
     7981 ctttggtgtc tattttctct tccataaaaa aagcctgact ccacttcccg cgtttactga
     8041 ttactagcga agctgcgggt gcattttttc aagataaagg catccccgat tatattctat
     8101 accgatgtgg attgcgcata ctttgtgaac agaaagtgat agcgttgatg attcttcatt
     8161 ggtcagaaaa ttatgaacgg tttcttctat tttgtctcta tatactacgt ataggaaatg
     8221 tttacatttt cgtattgttt tcgattcact ctatgaatag ttcttactac aatttttttg
     8281 tctaaagagt aatactagag ataaacataa aaaatgtaga ggtcgagttt agatgcaagt
     8341 tcaaggagcg aaaggtggat gggtaggtta tatagggata tagcacagag atatatagca
     8401 aagagatact tttgagcaat gtttgtggaa gcggtattcg caatatttta gtagctcgtt
     8461 acagtccggt gcgtttttgg ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa
     8521 aagcgctctg aagttcctat actttctaga gaataggaac ttcggaatag gaacttcaaa
     8581 gcgtttccga aaacgagcgc ttccgaaaat gcaacgcgag ctgcgcacat acagctcact
     8641 gttcacgtcg cacctatatc tgcgtgttgc ctgtatatat atatacatga gaagaacggc
     8701 atagtgcgtg tttatgctta aatgcgtact tatatgcgtc tatttatgta ggatgaaagg
     8761 tagtctagta cctcctgtga tattatccca ttccatgcgg ggtatcgtat gcttccttca
     8821 gcactaccct ttagctgttc tatatgctgc cactcctcaa ttggattagt ctcatccttc
     8881 aatgctatca tttcctttga tattggatca tactaagaaa ccattattat catgacatta
     8941 acctataaaa ataggcgtat cacgaggccc tttcgtc
//
