LOCUS       sequence-204901-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Atg42/Ybr139w(H474A) 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: pKP131 [pRS406-YBR139Wp-YBR139W(H474A)-GFP-ADH1t]
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     240..260
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2627..3337
                     /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(2654..2682)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3277..3297
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3361..3548
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3416..3440)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3569..3585)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3570..3586)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3610..3629)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3611..3629)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3639..3656)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3639..3655)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3648..3670)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3796..4251
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3883..3902)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4093..4114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4382..5185)
                     /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(5186..5401)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5425..5444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5544..5566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5604..5622)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5690..5794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5795..5863,5864..6655)
                     /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(6013..6032)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6826..7414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7315..7334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7568..7585
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggagctctt gcatggaggc gcaattggca
      301 aagacgtaaa gctcttgtca tcaattagat ctaatttgga aacagattgg tacaatttgg
      361 atggaatttt aggcttggca cctatcaatt gtgagcgacg ctttcttaca ttcaataact
      421 gttggttctg attttcactt tgactattgc cttggtatgg agaatttctc tgcaatggtt
      481 ttgcctgctt cgaaggagtg ctgttactat cgaagataaa ttccttcttt tcaaattgcc
      541 attgtaacat ggagttattc tctatatcct ggacgactgg aggagtcaag ggcctggaaa
      601 gtacggatcc tgtagaaata tcactggcaa ttatactgag tttattgttg gtgttgctat
      661 tcatatttga gctggtattg gacgaaagca aggataatga gtttgtgtct actgactcca
      721 acaccctagg ctggatttga tctttctcca tcctttaatt ttaacctttt aattagtggt
      781 tggatcaagt tttcgagact attccaatct gtgacttgtt ggataaatag tttttgactc
      841 gtttagtata atcctttttt ctaaaagtgc ttagagttct ctaagatgtt cttgtttaca
      901 atgtgagcga tttaggaaat ttcctaaaaa tggccgaggc ggcgctagca ttctacgaaa
      961 ggtgagataa cgcttcgtta tcgaaaaatg tcaggggaca ggggttatat aagaacgaaa
     1021 attgtcatcc tgcatttttt ctttaaaaca gctatacaaa aagtgatacc gacatacaat
     1081 gaagtatcta aacttagttt tcgtgcttca gcttcttatt agcatcaaat acgcctcatt
     1141 cggccgagcc ttttctcttt ttgaagatga taccaccttt gccaatttgg ataaacagct
     1201 aaagcttcca cagaatacac agcaaaccct taaattggac cgtttgaatc acgatgatcc
     1261 gctgtttaca acttttattt cttctgtgga cacagattac agtttgagac ttagaacagt
     1321 agatccttct aaactaggaa ttgacaccgt aaaacaatgg tcgggttaca tggactataa
     1381 ggattccaaa cacttttttt actggttttt tgaaagtagg aacgatcctg ctaacgaccc
     1441 aattattctt tggttaaatg gtggacctgg ttgttcctcg tttactgggt tgctatttga
     1501 actaggcccc tcatcaattg gcgccgatat gaaaccaatc cacaatccct attcttggaa
     1561 taataacgct tcaatgatct tcttagaaca gccactcgga gtcggctttt cctatggtga
     1621 tgaaaaagtc tcctctacaa aattagcagg caaagatgcg tacattttcc tggaattgtt
     1681 ttttgaagct tttcctcatt tacgctccaa cgatttccac attgcaggcg aatcctatgc
     1741 aggacattat atccctcaaa ttgcacatga gatcgttgtc aagaaccctg aaagaacgtt
     1801 caatttaact tcagttatga ttggtaatgg tatcacagac cctttgattc aagcagatta
     1861 ttatgaacca atggcatgcg ggaaaggggg ctatcaccct gttctctcat cagaagaatg
     1921 tgagaaaatg agtaaagctg caggtcgttg tcgtaggttg aacaagttat gttatgcttc
     1981 taaatcaagt ttaccatgca tagtcgccac tgcttactgt gactctgcac ttttggaacc
     2041 gtacattaac acaggactca acgtctatga cattagaggg ccctgtgaag ataatagtac
     2101 tgatggtatg tgttatacag gtctccgcta tgtcgaccag tatatgaatt ttcctgaagt
     2161 tcaagaaacg ctagggtccg acgtgcataa ttattctggc tgtgataatg acgtgttcac
     2221 cggatttttg tttacgggcg atggaagtaa accatttcaa caatatattg ctgaattatt
     2281 aaatcacaac attccggtat taatatatgc gggtgataag gattatattt gtaattggct
     2341 gggaaaccat gcttggtcca atgagttgga atggatcaat aaacgtaggt atcagagaag
     2401 gatgttaaga ccatgggtca gtaaagaaac aggtgaagag ttgggacaag tcaagaacta
     2461 tggccctttc acctttttga gaatatacga tgccggtgct atggtgccct atgatcaacc
     2521 ggaggcaagt ttggaaatgg tcaacagttg gatttccggt aatcgtgctt tttcggatct
     2581 ttccaccttg gaaaatgcta gttctagacg gatccccggg ctgcagatga aaggagaaga
     2641 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
     2701 attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt
     2761 tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcactta
     2821 tggtgttcaa tgcttttcaa gatacccaga tcatatgaaa cggcatgact ttttcaagag
     2881 tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta
     2941 caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa
     3001 aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa
     3061 ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa
     3121 aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac
     3181 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc
     3241 cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc
     3301 tgctgggatt acacatggca tggatgaact atacaaatag ggcgcgccac ttctaaataa
     3361 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta
     3421 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct
     3481 ttcctgtagg tcaggttgct ttctcaggta tagtatgagg tcgctcttat tgaccacacc
     3541 tctaccggga attcgatatc aagcttatcg ataccgtcga cctcgagggg gggcccggta
     3601 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     3661 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3721 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3781 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3841 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3901 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3961 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4021 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4081 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4141 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4201 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     4261 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagggtaat aactgatata
     4321 attaaattga agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt
     4381 tttagttttg ctggccgcat cttctcaaat atgcttccca gcctgctttt ctgtaacgtt
     4441 caccctctac cttagcatcc cttccctttg caaatagtcc tcttccaaca ataataatgt
     4501 cagatcctgt agagaccaca tcatccacgg ttctatactg ttgacccaat gcgtctccct
     4561 tgtcatctaa acccacaccg ggtgtcataa tcaaccaatc gtaaccttca tctcttccac
     4621 ccatgtctct ttgagcaata aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa
     4681 cagtaccctt agtatattct ccagtagata gggagccctt gcatgacaat tctgctaaca
     4741 tcaaaaggcc tctaggttcc tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa
     4801 tacctgggcc caccacaccg tgtgcattcg taatgtctgc ccattctgct attctgtata
     4861 cacccgcaga gtactgcaat ttgactgtat taccaatgtc agcaaatttt ctgtcttcga
     4921 agagtaaaaa attgtacttg gcggataatg cctttagcgg cttaactgtg ccctccatgg
     4981 aaaaatcagt caagatatcc acatgtgttt ttagtaaaca aattttggga cctaatgctt
     5041 caactaactc cagtaattcc ttggtggtac gaacatccaa tgaagcacac aagtttgttt
     5101 gcttttcgtg catgatatta aatagcttgg cagcaacagg actaggatga gtagcagcac
     5161 gttccttata tgtagctttc gacatgattt atcttcgttt cctgcaggtt tttgttctgt
     5221 gcagttgggt taagaatact gggcaatttc atgtttcttc aacactacat atgcgtatat
     5281 ataccaatct aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat
     5341 caaaaaaatt tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga
     5401 aaaggtggta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5461 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5521 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5581 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5641 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5701 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5761 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5821 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5881 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5941 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6001 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6061 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6121 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6181 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6241 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6301 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6361 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6421 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6481 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6541 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6601 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6661 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6721 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6781 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6841 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6901 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6961 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7021 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7081 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7141 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7201 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7261 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7321 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7381 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7441 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     7501 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     7561 cgagcgcagc gagtcagtga gcgaggaagc ggaag
//
