LOCUS       sequence-201590-        7606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Atg42/Ybr139w(D415A) 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 7606)
  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 7606)
  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: pKP133 [pRS406-YBR139Wp-YBR139W(D415A)-GFP-ADH1t]
FEATURES             Location/Qualifiers
     source          1..7606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2241..2261
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             4628..5338
                     /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(4655..4683)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5278..5298
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5362..5549
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5417..5441)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5570..5586)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5571..5587)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5611..5630)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5612..5630)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5640..5657)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5640..5656)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5649..5671)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5801..6256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5888..5907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6098..6119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6387..7190)
                     /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(7191..7411)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7437..7456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7556..7578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctct
     2281 tgcatggagg cgcaattggc aaagacgtaa agctcttgtc atcaattaga tctaatttgg
     2341 aaacagattg gtacaatttg gatggaattt taggcttggc acctatcaat tgtgagcgac
     2401 gctttcttac attcaataac tgttggttct gattttcact ttgactattg ccttggtatg
     2461 gagaatttct ctgcaatggt tttgcctgct tcgaaggagt gctgttacta tcgaagataa
     2521 attccttctt ttcaaattgc cattgtaaca tggagttatt ctctatatcc tggacgactg
     2581 gaggagtcaa gggcctggaa agtacggatc ctgtagaaat atcactggca attatactga
     2641 gtttattgtt ggtgttgcta ttcatatttg agctggtatt ggacgaaagc aaggataatg
     2701 agtttgtgtc tactgactcc aacaccctag gctggatttg atctttctcc atcctttaat
     2761 tttaaccttt taattagtgg ttggatcaag ttttcgagac tattccaatc tgtgacttgt
     2821 tggataaata gtttttgact cgtttagtat aatccttttt tctaaaagtg cttagagttc
     2881 tctaagatgt tcttgtttac aatgtgagcg atttaggaaa tttcctaaaa atggccgagg
     2941 cggcgctagc attctacgaa aggtgagata acgcttcgtt atcgaaaaat gtcaggggac
     3001 aggggttata taagaacgaa aattgtcatc ctgcattttt tctttaaaac agctatacaa
     3061 aaagtgatac cgacatacaa tgaagtatct aaacttagtt ttcgtgcttc agcttcttat
     3121 tagcatcaaa tacgcctcat tcggccgagc cttttctctt tttgaagatg ataccacctt
     3181 tgccaatttg gataaacagc taaagcttcc acagaataca cagcaaaccc ttaaattgga
     3241 ccgtttgaat cacgatgatc cgctgtttac aacttttatt tcttctgtgg acacagatta
     3301 cagtttgaga cttagaacag tagatccttc taaactagga attgacaccg taaaacaatg
     3361 gtcgggttac atggactata aggattccaa acactttttt tactggtttt ttgaaagtag
     3421 gaacgatcct gctaacgacc caattattct ttggttaaat ggtggacctg gttgttcctc
     3481 gtttactggg ttgctatttg aactaggccc ctcatcaatt ggcgccgata tgaaaccaat
     3541 ccacaatccc tattcttgga ataataacgc ttcaatgatc ttcttagaac agccactcgg
     3601 agtcggcttt tcctatggtg atgaaaaagt ctcctctaca aaattagcag gcaaagatgc
     3661 gtacattttc ctggaattgt tttttgaagc ttttcctcat ttacgctcca acgatttcca
     3721 cattgcaggc gaatcctatg caggacatta tatccctcaa attgcacatg agatcgttgt
     3781 caagaaccct gaaagaacgt tcaatttaac ttcagttatg attggtaatg gtatcacaga
     3841 ccctttgatt caagcagatt attatgaacc aatggcatgc gggaaagggg gctatcaccc
     3901 tgttctctca tcagaagaat gtgagaaaat gagtaaagct gcaggtcgtt gtcgtaggtt
     3961 gaacaagtta tgttatgctt ctaaatcaag tttaccatgc atagtcgcca ctgcttactg
     4021 tgactctgca cttttggaac cgtacattaa cacaggactc aacgtctatg acattagagg
     4081 gccctgtgaa gataatagta ctgatggtat gtgttataca ggtctccgct atgtcgacca
     4141 gtatatgaat tttcctgaag ttcaagaaac gctagggtcc gacgtgcata attattctgg
     4201 ctgtgataat gacgtgttca ccggattttt gtttacgggc gatggaagta aaccatttca
     4261 acaatatatt gctgaattat taaatcacaa cattccggta ttaatatatg cgggtgataa
     4321 ggcttatatt tgtaattggc tgggaaacca tgcttggtcc aatgagttgg aatggatcaa
     4381 taaacgtagg tatcagagaa ggatgttaag accatgggtc agtaaagaaa caggtgaaga
     4441 gttgggacaa gtcaagaact atggcccttt cacctttttg agaatatacg atgccggtca
     4501 tatggtgccc tatgatcaac cggaggcaag tttggaaatg gtcaacagtt ggatttccgg
     4561 taatcgtgct ttttcggatc tttccacctt ggaaaatgct agttctagac ggatccccgg
     4621 gctgcagatg aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
     4681 tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata
     4741 cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac
     4801 acttgtcact actttcactt atggtgttca atgcttttca agatacccag atcatatgaa
     4861 acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt
     4921 tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct
     4981 tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca
     5041 caaattggaa tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa
     5101 tggaatcaaa gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc
     5161 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
     5221 ttacctgtcc acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt
     5281 ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata
     5341 gggcgcgcca cttctaaata agcgaatttc ttatgattta tgatttttat tattaaataa
     5401 gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt aaaacgaaaa
     5461 ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt atagtatgag
     5521 gtcgctctta ttgaccacac ctctaccggg aattcgatat caagcttatc gataccgtcg
     5581 acctcgaggg ggggcccggt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     5641 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     5701 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     5761 ccttcccaac agttgcgcag cctgaatggc gaatggcgcg acgcgccctg tagcggcgca
     5821 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5881 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5941 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6001 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     6061 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     6121 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     6181 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     6241 ttaacgttta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     6301 accgcatagg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata
     6361 catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct
     6421 tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat
     6481 agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta
     6541 tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac
     6601 caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa
     6661 tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag
     6721 cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct
     6781 gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg
     6841 tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca
     6901 atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt
     6961 agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt
     7021 aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca
     7081 tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca
     7141 acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt
     7201 cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt
     7261 tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct
     7321 tccttctgtt cggagattac cgaatcaaaa aaatttcaaa gaaaccgaaa tcaaaaaaaa
     7381 gaataaaaaa aaaatgatga attgaattga aaagctgtgg tatggtgcac tctcagtaca
     7441 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     7501 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     7561 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcga
//
