LOCUS       sequence-56699-e        6878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast expression of VPH2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6878)
  AUTHORS   Hughes AL, Gottschling DE
  TITLE     An early age increase in vacuolar pH limits mitochondrial function 
            and lifespan in yeast.
  JOURNAL   Nature. 2012 Dec 13;492(7428):261-5. doi: 10.1038/nature11654. Epub 
            2012 Nov 21.
  PUBMED    23172144
REFERENCE   2  (bases 1 to 6878)
  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: 
            "10.1038/nature11654"; journalName: "Nature"; date: "2012-12-13- 
            13"; volume: "492"; issue: "7428"; pages: "261-5"
COMMENT     Sequence Label: pAG306-GPD-VPH2 chr I
FEATURES             Location/Qualifiers
     source          1..6878
                     /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        190..410
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             411..1214
                     /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(1343..1798)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1480..1501)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1692..1711
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1928..1950
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1942..1959
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1943..1959
                     /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"
     promoter        2009..2653
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     2547..2570
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     2659..2675
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2659..2675
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2683..2707
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3362..3386)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3419..3435)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3420..3436)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      3438..3685
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3454..3472)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(3708..3726)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3747..3763)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3747..3763)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3760..3782)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3771..3787
                     /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(3795..3801,3802..3819,3820..3825))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3978..3995)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4149..4737)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4908..5699,5700..5768))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5531..5550
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5769..5873)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6851..6869
                     /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 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc
      241 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca
      301 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat
      361 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag
      421 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata
      481 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat
      541 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata
      601 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt
      661 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc
      721 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg
      781 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac
      841 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat
      901 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg
      961 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg
     1021 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg
     1081 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg
     1141 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg
     1201 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta
     1261 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt
     1321 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta
     1381 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     1441 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     1501 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     1561 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     1621 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     1681 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     1741 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     1801 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     1861 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca
     1921 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
     1981 tagggcgaat tggagctctt cgagtttatc attatcaata ctcgccattt caaagaatac
     2041 gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt agccttttaa
     2101 ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat atataacacc
     2161 gaaggtgcct gggtgaacag gtttattcct ggcatccact aaatataatg gagcccgctt
     2221 tttaagctgg catccagaaa aaaaaagaat cccagcacca aaatattgtt ttcttcacca
     2281 accatcagtt cataggtcca ttctcttagc gcaactacag agaacagggg cacaaacagg
     2341 caaaaaacgg gcacaacctc aatggagtga tgcaacctgc ctggagtaaa tgatgacaca
     2401 aggcaattga cccacgcatg tatctatctc attttcttac accttctatt accttctgct
     2461 ctctctgatt tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga aattattccc
     2521 ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg taaatctatt
     2581 tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt aaaacaccag
     2641 aacttagttt cgacggattc tagaactagt ggatccccca tcacaagttt gtacaaaaaa
     2701 gcaggctcca ccatgttcga aattaaactg aatgatagaa taactgaatt tctgcgaaag
     2761 ttcaagaata gcgcgaaatc aaacgaagga attgatgagg acattgattt gttcctgaag
     2821 cgacacgcaa tacctatgca gtcacttctt ttttatgtga aagagtatag aaaagacagt
     2881 gatctccaat gctctatcaa agaacttttg aaacctttgg agttcgaatt caagcccaag
     2941 gccgtcagag gtttacacta ttcagaggat ttcaagaaaa aattagagtt tttgaaatat
     3001 caggaacaag aacttgaata tcaaagtatg gtcaagagaa gcaagtctgt cttttcactt
     3061 caagaagacg atgaactcac gccttcacaa attaataaac agattaagga acaagttacc
     3121 actgttttca acgttcttgt aagtgtcata tcggttgttg tggccatttg gtactggacc
     3181 ggatcttcaa caaactttcc ggtccatgtt cgtcttttgt tgtgcttgtt ctttggaata
     3241 ttggtcttag tagcagatgt ggttgtgtat aacagctatc tgaaaaaact cgaagaagca
     3301 aaagtaaaag aaaaaacaaa ggttgagaaa aagaaagttc taagcaagat tacactgtag
     3361 gacccagctt tcttgtacaa agtggtgatg ggctgcagga attcgatatc aagcttatcg
     3421 ataccgtcga cctcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc
     3481 cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg tccctattta
     3541 tttttttata gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt
     3601 tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg agaaggtttt
     3661 gggacgctcg aaggctttaa tttgcggccg gtacccagct tttgttccct ttagtgaggg
     3721 ttaattccga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3781 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa
     3841 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3901 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3961 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4021 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4081 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4141 ctggcgtttt tccataggct cggcccccct gacgagcatc acaaaaatcg acgctcaagt
     4201 cagaggtggc gaaacccgac aggactataa agataccagg cgttcccccc tggaagctcc
     4261 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4321 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     4381 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4441 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4501 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4561 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4621 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4681 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4741 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4801 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4861 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4921 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactg
     4981 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5041 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5101 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5161 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5221 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5281 caacgatcaa ggcgagttac atgatccccc atgttgtgaa aaaaagcggt tagctccttc
     5341 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5401 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     5461 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     5521 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     5581 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5641 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5701 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5761 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5821 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5881 ccccgaaaag tgccacctgg ggggtgctaa ttatggcatt gattggggcg gtaaaacatt
     5941 caaagtcaat gaaaaggatg ctaaattgaa tgatactgaa gacatttcat tcgcttctca
     6001 gatcaaaaac ttaaatctgt ctatatgagc tgtcccaaag ccacttctta cctttcaaga
     6061 aaacatggac cttgctttgc cacaaaataa ttgcctgtaa gatagttttt tatttgcaca
     6121 ttgttattac taacctatgc tttgctacgg acgactgctt ggtaaaaatg caaaagtatt
     6181 aggatgtctc acaagaaact gctcgtggtg aaaattttca gccgcgacca tcctcccacg
     6241 ccaacaagcc ctcttttgcc catccttctg gaattcgcgg tgaaaatcaa tttgatatat
     6301 aaaattccct cataatatct aggaacggga tcacatatct caatgagcta aagcggccgc
     6361 ttcttcgaat gggaccagct aaagtgatca aacttcatgc aggatgttgg acactttttc
     6421 tttcaagaag ctccaaatat ataaattttt gcaatatagg aattgaaata tcgtacttcc
     6481 gattcagaat atctcaactc tactacatct atatatgact gtactcgcgt tcttggtaaa
     6541 catcaattag taggtgtttt tgcgagcata tgttgagctc agaaaatatg agcacaatct
     6601 tcgaaaccat tgtttttttg tgcaaaagtc accatcgggt ttatagtatg gactcttgaa
     6661 cgaaccattc tgatctgtat ttctctcata tttactccat ttattttttg ctgctctttg
     6721 ctttacttca acacgatgct tttcctaagt aattttttat tttattcaca atgctccata
     6781 actcttcagg ccttaaacga ccatccatgg cccctaagaa accattatta tcatgacatt
     6841 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
