LOCUS       sequence-56696-e        7879 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 7879)
  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 7879)
  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-ccdB chr I
FEATURES             Location/Qualifiers
     source          1..7879
                     /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..2807
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2832..2837,2838..2855,2856..2862)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2916..3575
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2982..3001)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3917..4222
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4101..4120
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4263..4387)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4420..4436)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4421..4437)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     terminator      4439..4686
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4455..4473)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(4709..4727)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4748..4764)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4748..4764)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4761..4783)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4772..4788
                     /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(4796..4802,4803..4820,4821..4826))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4979..4996)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5150..5738)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5909..6700,6701..6769))
                     /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     6532..6551
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6770..6874)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7852..7870
                     /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 gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa
     2761 aaacagacta cataatactg taaaacacaa catatccagt cactatggcg gccgcattag
     2821 gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg
     2881 atccgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata
     2941 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
     3001 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa
     3061 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg
     3121 ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc
     3181 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat
     3241 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
     3301 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc
     3361 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc
     3421 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga
     3481 ttcaggttca tcatgccgtc tgtgatggct tccatgtcgg cagaatgctt aatgaattac
     3541 aacagtactg cgatgagtgg cagggcgggg cgtaaacgcg tggatccggc ttactaaaag
     3601 ccagataaca gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata
     3661 tgtatacccg aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt
     3721 tgacagcgac agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta
     3781 agcacaacca tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat
     3841 caggaaggga tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag
     3901 aacaggggct ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     3961 tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc
     4021 cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     4081 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     4141 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     4201 cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg
     4261 accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
     4321 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct ttcttgtaca
     4381 aagtggtgat gggctgcagg aattcgatat caagcttatc gataccgtcg acctcgagtc
     4441 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa
     4501 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
     4561 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac
     4621 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta
     4681 atttgcggcc ggtacccagc ttttgttccc tttagtgagg gttaattccg agcttggcgt
     4741 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4801 taggagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagg taactcacat
     4861 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4921 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4981 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5041 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5101 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5161 tcggcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5221 caggactata aagataccag gcgttccccc ctggaagctc cctcgtgcgc tctcctgttc
     5281 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5341 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5401 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5461 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     5521 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     5581 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5641 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5701 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5761 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5821 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5881 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5941 cagcgatctg tctatttcgt tcatccatag ttgcctgact gcccgtcgtg tagataacta
     6001 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6061 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6121 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6181 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6241 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6301 catgatcccc catgttgtga aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6361 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6421 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6481 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     6541 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6601 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6661 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6721 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6781 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6841 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6901 gggggtgcta attatggcat tgattggggc ggtaaaacat tcaaagtcaa tgaaaaggat
     6961 gctaaattga atgatactga agacatttca ttcgcttctc agatcaaaaa cttaaatctg
     7021 tctatatgag ctgtcccaaa gccacttctt acctttcaag aaaacatgga ccttgctttg
     7081 ccacaaaata attgcctgta agatagtttt ttatttgcac attgttatta ctaacctatg
     7141 ctttgctacg gacgactgct tggtaaaaat gcaaaagtat taggatgtct cacaagaaac
     7201 tgctcgtggt gaaaattttc agccgcgacc atcctcccac gccaacaagc cctcttttgc
     7261 ccatccttct ggaattcgcg gtgaaaatca atttgatata taaaattccc tcataatatc
     7321 taggaacggg atcacatatc tcaatgagct aaagcggccg cttcttcgaa tgggaccagc
     7381 taaagtgatc aaacttcatg caggatgttg gacacttttt ctttcaagaa gctccaaata
     7441 tataaatttt tgcaatatag gaattgaaat atcgtacttc cgattcagaa tatctcaact
     7501 ctactacatc tatatatgac tgtactcgcg ttcttggtaa acatcaatta gtaggtgttt
     7561 ttgcgagcat atgttgagct cagaaaatat gagcacaatc ttcgaaacca ttgttttttt
     7621 gtgcaaaagt caccatcggg tttatagtat ggactcttga acgaaccatt ctgatctgta
     7681 tttctctcat atttactcca tttatttttt gctgctcttt gctttacttc aacacgatgc
     7741 ttttcctaag taatttttta ttttattcac aatgctccat aactcttcag gccttaaacg
     7801 accatccatg gcccctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     7861 atcacgaggc cctttcgtc
//
