LOCUS       sequence-472498-        7955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of FL ScVPS34-3xFLAG under its own promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7955)
  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 7955)
  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: pYO478
FEATURES             Location/Qualifiers
     source          1..7955
                     /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        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1344..1799)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1481..1502)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1693..1712
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1925..1947
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1939..1956
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1940..1956
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1966..1985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1966..1984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2187..2198
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4844..4909
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      4926..5113
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4981..5005)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5182..5202)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5182..5200)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5221..5237)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5221..5237)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5234..5256)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5245..5261
                     /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(5269..5275,5276..5293,5294..5299))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5314..5335
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5452..5469)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5623..6211)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5703..5722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6382..7173,7174..7242))
                     /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     7005..7024
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7243..7347)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7384..7887
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     7928..7946
                     /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 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt
     1081 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctatgcggtg tgaaataccg cacagatgcg
     1321 taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa aattcgcgtt
     1381 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     1441 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     1501 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     1561 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     1621 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     1681 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     1741 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     1801 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     1861 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     1921 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     1981 tagggcgaat tgggtaccgg ggctgaagct ttcgacaaat cttaaattgt gactttgtgc
     2041 atgaaatttt tatatgaacg ttttaaataa gtggacatag tccttcattc ggtatatgca
     2101 gatccggcat caaacacttt ttcaagaaag tttgagtcaa aaggattaaa ggataaagta
     2161 aaacaacaat aacatctccg tgaagcattg agggaagggt ttaactccaa caatgtcact
     2221 gaacaacata acattctgtg tctcacagga tctggatgtt cccctgaaag tgaaaatcaa
     2281 gtcattggaa ggacataagc cactgttgaa gccatctcaa aaaatcctga accctgaatt
     2341 aatgctgata gggtcaaatg tattcccttc tagtgatcta attgtatcct tacaggtatt
     2401 tgataaagag agaaacagaa atttaactct tccgatatac acgccgtata ttcctttcag
     2461 aaactctaga acctgggatt attggttgac tttgcccata cgtatcaaac agcttacctt
     2521 tagcagtcat ttacgcatta ttttgtggga atacaatgga tccaagcaaa ttcccttttt
     2581 caatctagaa acgagcatct ttaacttaaa agactgtact ttaaaaagag ggtttgaatc
     2641 cttaaaattt cgttatgatg tgattgacca ctgtgaagtg gtcactgata acaaagatca
     2701 agaaaatttg aataaatatt ttcagggtga attcacgaga ctcccttggc ttgacgaaat
     2761 cactataagc aaattaagga aacaacggga aaataggact tggcctcagg gcacctttgt
     2821 cttaaactta gaatttccaa tgttagagct tcctgttgtg ttcatcgaaa gggaaattat
     2881 gaatactcaa atgaatattc caactttgaa gaacaatcct ggactaagca cagacttacg
     2941 agaaccgaat aggaatgatc ctcaaatcaa aatttctttg ggggacaaat atcactccac
     3001 attgaagttt tacgatcctg accaaccaaa caatgatcca atagaggaaa agtataggag
     3061 attggaaaga gcatctaaaa atgcaaactt ggacaagcaa gtgaagccgg acattaaaaa
     3121 gagggactac ttgaataaaa tcatcaacta cccccctggc actaaattaa cagcacatga
     3181 aaagggttca atatggaaat ataggtatta tttgatgaac aataaaaaag ctcttacaaa
     3241 gttattacag agtacaaatt tgagagaaga atcggaaaga gtagaggttt tagaactaat
     3301 ggactcatgg gcagaaattg atatagacga cgcattagaa ctattaggct caacttttaa
     3361 aaacctttca gtgagatcgt acgccgtaaa taggctgaag aaggcatcag acaaggagct
     3421 ggaattgtac ttattacagt tggtagaagc tgtctgtttt gagaacctgt ccactttttc
     3481 cgacaaatct aacagtgaat tcactattgt ggatgccgta tcttcgcaaa agctttcggg
     3541 tgattctatg ttactatcta catcgcatgc caaccaaaaa ttattaaagt ccatctcgag
     3601 tgaatcggaa acttccggga cagaatcgct accaatcgtc atttctccct tggctgagtt
     3661 tttgattagg agagcactgg tcaacccaag gttaggaagt tttttctatt ggtatttaaa
     3721 gtccgagtct gaggacaagc catatttaga ccagatttta agttcctttt ggagtagact
     3781 ggataaaaaa tctcggaata tactgaacga tcaagtcagg ctaataaatg tgctcaggga
     3841 atgttgtgaa acaattaaga ggctgaaaga tactacagcc aaaaagatgg aattactagt
     3901 gcatttattg gagacgaaag tcaggcctct tgtcaaagta cgaccaattg ctttaccact
     3961 agaccctgat gtgttgatat gcgacgtttg tcctgaaact tcgaaggtat ttaaaagctc
     4021 tttatcgcca ctaaaaataa cattcaaaac gaccttaaac caaccatatc acttaatgtt
     4081 taaggttggc gatgatttga gacaagatca actggtagta cagatcataa gtttaatgaa
     4141 tgagttatta aaaaatgaaa acgtggactt gaaattgaca ccatacaaaa ttttggcaac
     4201 aggaccacaa gaaggtgcca ttgaatttat ccctaatgac acattagcta gcatactaag
     4261 caaatatcac ggtattcttg gctaccttaa actccactat cctgatgaga acgcgacatt
     4321 aggcgtacaa ggctgggttt tagataattt tgtcaaatct tgtgctggct attgtgttat
     4381 tacatacatc ttaggtgtcg gcgataggca tttagacaac ttactagtca cgccagatgg
     4441 gcactttttt cacgcagact ttggttatat cttgggtcag gaccccaaac cttttccgcc
     4501 attaatgaaa ttgccccctc aaattataga ggcgtttggg ggtgcagaat catcaaatta
     4561 tgataaattt cgcagctact gttttgtggc atattcgatt ttaagaagga atgcaggctt
     4621 aatcttaaac ctgtttgaat taatgaagac ttcgaacata cctgatatca gaatagatcc
     4681 caatggtgct atattacgtg taagagagag atttaatttg aatatgtccg aagaagatgc
     4741 cacagtgcat tttcagaatc taatcaatga tagtgtaaat gctttgctgc ctatcgtgat
     4801 tgatcattta cataatctgg cacaatactg gcggaccgtc gaggactaca aagaccatga
     4861 cggtgattat aaagatcatg atatcgatta caaggatgac gatgacaagt agaattcgtg
     4921 agctcgcgaa tttcttatga tttatgattt ttattattaa ataagttata aaaaaaataa
     4981 gtgtatacaa attttaaagt gactcttagg ttttaaaacg aaaattctta ttcttgagta
     5041 actctttcct gtaggtcagg ttgctttctc aggtatagca tgaggtcgct cttattgacc
     5101 acacctctac cggcatgccg agcaaatgcc tgcaaatcgc tccccgcggc cgccaccgcg
     5161 gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg
     5221 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5281 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5341 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5401 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5461 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5521 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5581 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5641 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5701 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5761 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5821 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5881 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5941 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6001 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6061 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6121 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6181 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6241 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6301 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6361 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6421 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6481 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6541 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6601 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6661 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6721 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6781 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6841 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6901 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6961 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     7021 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7081 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7141 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7201 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7261 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7321 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcctt
     7381 ttcatcacgt gctataaaaa taattataat ttaaattttt taatataaat atataaatta
     7441 aaaatagaaa gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc gaagatgtaa
     7501 aagactctag ggggatcgcc aacaaatact accttttatc ttgctcttcc tgctctcagg
     7561 tattaatgcc gaattgtttc atcttgtctg tgtagaagac cacacacgaa aatcctgtga
     7621 ttttacattt tacttatcgt taatcgaatg tatatctatt taatctgctt ttcttgtcta
     7681 ataaatatat atgtaaagta cgctttttgt tgaaattttt taaacctttg tttatttttt
     7741 tttcttcatt ccgtaactct tctaccttct ttatttactt tctaaaatcc aaatacaaaa
     7801 cataaaaata aataaacaca gagtaaattc ccaaattatt ccatcattaa aagatacgag
     7861 gcgcgtgtaa gttacaggca agcgatccgt ctaagaaacc attattatca tgacattaac
     7921 ctataaaaat aggcgtatca cgaggccctt tcgtc
//
