LOCUS       sequence-204900-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Atg42/Ybr139w with a C-terminal GFP tag in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7595)
  AUTHORS   Parzych KR, Ariosa A, Mari M, Klionsky DJ
  TITLE     A newly characterized vacuolar serine carboxypeptidase, 
            Atg42/Ybr139w, is required for normal vacuole function and the 
            terminal steps of autophagy in the yeast Saccharomyces cerevisiae.
  JOURNAL   Mol Biol Cell. 2018 May 1;29(9):1089-1099. doi: 
            10.1091/mbc.E17-08-0516. Epub 2018 Mar 22.
  PUBMED    29514932
REFERENCE   2  (bases 1 to 7595)
  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: 
            "10.1091/mbc.E17-08-0516"; journalName: "Mol Biol Cell"; date: 
            "2018-05-1- 1"; volume: "29"; issue: "9"; pages: "1089-1099"
COMMENT     Sequence Label: pKP115 (pRS406-YBR139Wp-YBR139W-GFP-ADH1t)
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    97..118
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(133..138,139..156,157..163)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    171..187
                     /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)."
     primer_bind     176..198
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     195..211
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     195..211
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     230..250
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        232..250
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2617..3327
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2644..2672)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3267..3287
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3351..3538
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3406..3430)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3559..3575)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3560..3576)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3600..3619)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3601..3619)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3629..3646)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3629..3645)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3638..3660)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3786..4241
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3873..3892)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4083..4104
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4372..5175)
                     /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"
     promoter        complement(5176..5391)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5415..5434)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5534..5556
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5594..5612)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5680..5784
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5785..5853,5854..6645)
                     /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     complement(6003..6022)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6816..7404
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7305..7324
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7558..7575
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
       61 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      121 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      181 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
      241 tcactaaagg gaacaaaagc tggagctctt gcatggaggc gcaattggca aagacgtaaa
      301 gctcttgtca tcaattagat ctaatttgga aacagattgg tacaatttgg atggaatttt
      361 aggcttggca cctatcaatt gtgagcgacg ctttcttaca ttcaataact gttggttctg
      421 attttcactt tgactattgc cttggtatgg agaatttctc tgcaatggtt ttgcctgctt
      481 cgaaggagtg ctgttactat cgaagataaa ttccttcttt tcaaattgcc attgtaacat
      541 ggagttattc tctatatcct ggacgactgg aggagtcaag ggcctggaaa gtacggatcc
      601 tgtagaaata tcactggcaa ttatactgag tttattgttg gtgttgctat tcatatttga
      661 gctggtattg gacgaaagca aggataatga gtttgtgtct actgactcca acaccctagg
      721 ctggatttga tctttctcca tcctttaatt ttaacctttt aattagtggt tggatcaagt
      781 tttcgagact attccaatct gtgacttgtt ggataaatag tttttgactc gtttagtata
      841 atcctttttt ctaaaagtgc ttagagttct ctaagatgtt cttgtttaca atgtgagcga
      901 tttaggaaat ttcctaaaaa tggccgaggc ggcgctagca ttctacgaaa ggtgagataa
      961 cgcttcgtta tcgaaaaatg tcaggggaca ggggttatat aagaacgaaa attgtcatcc
     1021 tgcatttttt ctttaaaaca gctatacaaa aagtgatacc gacatacaat gaagtatcta
     1081 aacttagttt tcgtgcttca gcttcttatt agcatcaaat acgcctcatt cggccgagcc
     1141 ttttctcttt ttgaagatga taccaccttt gccaatttgg ataaacagct aaagcttcca
     1201 cagaatacac agcaaaccct taaattggac cgtttgaatc acgatgatcc gctgtttaca
     1261 acttttattt cttctgtgga cacagattac agtttgagac ttagaacagt agatccttct
     1321 aaactaggaa ttgacaccgt aaaacaatgg tcgggttaca tggactataa ggattccaaa
     1381 cacttttttt actggttttt tgaaagtagg aacgatcctg ctaacgaccc aattattctt
     1441 tggttaaatg gtggacctgg ttgttcctcg tttactgggt tgctatttga actaggcccc
     1501 tcatcaattg gcgccgatat gaaaccaatc cacaatccct attcttggaa taataacgct
     1561 tcaatgatct tcttagaaca gccactcgga gtcggctttt cctatggtga tgaaaaagtc
     1621 tcctctacaa aattagcagg caaagatgcg tacattttcc tggaattgtt ttttgaagct
     1681 tttcctcatt tacgctccaa cgatttccac attgcaggcg aatcctatgc aggacattat
     1741 atccctcaaa ttgcacatga gatcgttgtc aagaaccctg aaagaacgtt caatttaact
     1801 tcagttatga ttggtaatgg tatcacagac cctttgattc aagcagatta ttatgaacca
     1861 atggcatgcg ggaaaggggg ctatcaccct gttctctcat cagaagaatg tgagaaaatg
     1921 agtaaagctg caggtcgttg tcgtaggttg aacaagttat gttatgcttc taaatcaagt
     1981 ttaccatgca tagtcgccac tgcttactgt gactctgcac ttttggaacc gtacattaac
     2041 acaggactca acgtctatga cattagaggg ccctgtgaag ataatagtac tgatggtatg
     2101 tgttatacag gtctccgcta tgtcgaccag tatatgaatt ttcctgaagt tcaagaaacg
     2161 ctagggtccg acgtgcataa ttattctggc tgtgataatg acgtgttcac cggatttttg
     2221 tttacgggcg atggaagtaa accatttcaa caatatattg ctgaattatt aaatcacaac
     2281 attccggtat taatatatgc gggtgataag gattatattt gtaattggct gggaaaccat
     2341 gcttggtcca atgagttgga atggatcaat aaacgtaggt atcagagaag gatgttaaga
     2401 ccatgggtca gtaaagaaac aggtgaagag ttgggacaag tcaagaacta tggccctttc
     2461 acctttttga gaatatacga tgccggtcat atggtgccct atgatcaacc ggaggcaagt
     2521 ttggaaatgg tcaacagttg gatttccggt aatcgtgctt tttcggatct ttccaccttg
     2581 gaaaatgcta gttctagacg gatccccggg ctgcagatga aaggagaaga acttttcact
     2641 ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc
     2701 agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt tatttgcact
     2761 actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcactta tggtgttcaa
     2821 tgcttttcaa gatacccaga tcatatgaaa cggcatgact ttttcaagag tgccatgccc
     2881 gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta caagacacgt
     2941 gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgat
     3001 tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa ctcacacaat
     3061 gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa aattagacac
     3121 aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc
     3181 gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa
     3241 gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt
     3301 acacatggca tggatgaact atacaaatag ggcgcgccac ttctaaataa gcgaatttct
     3361 tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt
     3421 aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg
     3481 tcaggttgct ttctcaggta tagtatgagg tcgctcttat tgaccacacc tctaccggga
     3541 attcgatatc aagcttatcg ataccgtcga cctcgagggg gggcccggta cccaattcgc
     3601 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg
     3661 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3721 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3781 aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3841 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3901 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3961 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     4021 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4081 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     4141 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     4201 atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg gtattttctc
     4261 cttacgcatc tgtgcggtat ttcacaccgc atagggtaat aactgatata attaaattga
     4321 agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt tttagttttg
     4381 ctggccgcat cttctcaaat atgcttccca gcctgctttt ctgtaacgtt caccctctac
     4441 cttagcatcc cttccctttg caaatagtcc tcttccaaca ataataatgt cagatcctgt
     4501 agagaccaca tcatccacgg ttctatactg ttgacccaat gcgtctccct tgtcatctaa
     4561 acccacaccg ggtgtcataa tcaaccaatc gtaaccttca tctcttccac ccatgtctct
     4621 ttgagcaata aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt
     4681 agtatattct ccagtagata gggagccctt gcatgacaat tctgctaaca tcaaaaggcc
     4741 tctaggttcc tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc
     4801 caccacaccg tgtgcattcg taatgtctgc ccattctgct attctgtata cacccgcaga
     4861 gtactgcaat ttgactgtat taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa
     4921 attgtacttg gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt
     4981 caagatatcc acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc
     5041 cagtaattcc ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg
     5101 catgatatta aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata
     5161 tgtagctttc gacatgattt atcttcgttt cctgcaggtt tttgttctgt gcagttgggt
     5221 taagaatact gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct
     5281 aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt
     5341 tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaaggtggta
     5401 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5461 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5521 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5581 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5641 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5701 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5761 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5821 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5881 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5941 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6001 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6061 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6121 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6181 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6241 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6301 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6361 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6421 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6481 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6541 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6601 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6661 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6721 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6781 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6841 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6901 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6961 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7021 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7081 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7141 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7201 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7261 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7321 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7381 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7441 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7501 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7561 gagtcagtga gcgaggaagc ggaagagcgc ccaat
//
