LOCUS       sequence-56701-e        4894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4894)
  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 4894)
  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: pKT127-SEP
FEATURES             Location/Qualifiers
     source          1..4894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..62)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     162..181
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        262..280
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     262..279
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             350..1066
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(380..408)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1003..1023
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1087..1274
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1142..1166)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1349..2705
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        1349..1692
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1693..2502
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1760..1779)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2508..2705
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2809..2828)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2810..2828)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2915..2932)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3086..3674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3166..3185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3845..4636,4637..4705))
                     /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     4468..4487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4706..4810)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(4878..4894,1..2)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
       61 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
      121 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
      181 actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt aatacataac
      241 cttatgtatc atacacatac gatttaggtg acactataga acgcggccgc cagctgaagc
      301 ttcgtacgct gcaggtcgac ggatcggtga cggtgctggt ttaattaaca tgagtaaagg
      361 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
      421 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
      481 taaatttatt tgcactactg gaaaactacc tgttccttgg ccaacacttg tcactacttt
      541 aacttatggt gttcaatgct tttcaagata cccagatcat atgaaacggc atgacttttt
      601 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
      661 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
      721 gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa
      781 ctataacgat caccaggtgt acatcatggc agacaaacaa aagaatggaa tcaaagctaa
      841 cttcaaaatt agacacaaca ttgaagatgg aggcgttcaa ctagcagacc attatcaaca
      901 aaatactcca attggcgatg ggcccgtcct tttaccagac aaccattacc tgtttacaac
      961 ttctactctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
     1021 aacagctgct gggattacac atggcatgga tgaactatac aaataaggcg cgccacttct
     1081 aaataagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     1141 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt
     1201 aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac
     1261 cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatctg tttagcttgc
     1321 ctcgtccccg ccgggtcacc cggccagcga catggaggcc cagaataccc tccttgacag
     1381 tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt agcccataca
     1441 tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc gaagcaaaaa
     1501 ttacggctcc tcgctgcaga cctgcgagca gggaaacgct cccctcacag acgcgttgaa
     1561 ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc cactgaggtt
     1621 cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca tcacatccga
     1681 acataaacaa ccatgggtaa ggaaaagact cacgtttcga ggccgcgatt aaattccaac
     1741 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     1801 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     1861 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     1921 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     1981 actgcgatcc ccggcaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     2041 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     2101 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     2161 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     2221 tggaaagaaa tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     2281 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     2341 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     2401 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     2461 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagtact gacaataaaa
     2521 agattcttgt tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat
     2581 tttaatcaaa tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg
     2641 cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta
     2701 tactgctgtc gattcgatac taacgccgcc atccagttta aacgagctcg aattcatcga
     2761 tgatatcaga tccactagtg gcctatgcgg ccgcggatct gccggtctcc ctatagtgag
     2821 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
     2881 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2941 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3001 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3061 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3121 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3181 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3241 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     3301 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3361 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3421 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3481 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3541 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3601 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3661 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3721 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3781 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3841 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3901 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3961 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4021 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4081 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4141 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4201 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4261 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4321 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4381 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4441 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4501 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4561 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4621 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4681 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4741 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4801 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     4861 tgacattaac ctataaaaat aggcgtatca cgag
//
