LOCUS       sequence-204903-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Atg42/Ybr139w(S219,D415,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: pKP134
            [pRS406-YBR139Wp-YBR139W(S219,D415,H474...
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    109..130
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(145..150,151..168,169..175)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    183..199
                     /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     188..210
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     207..223
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     207..223
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     242..262
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        244..262
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2629..3339
                     /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(2656..2684)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3279..3299
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3363..3550
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3418..3442)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3571..3587)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3572..3588)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3612..3631)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3613..3631)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3641..3658)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3641..3657)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3650..3672)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3798..4253
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3885..3904)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4095..4116
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4384..5187)
                     /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(5188..5403)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5427..5446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5546..5568
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5606..5624)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5692..5796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5797..5865,5866..6657)
                     /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(6015..6034)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6828..7416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7317..7336
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7570..7587
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
       61 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      121 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      181 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
      241 cgcaattaac cctcactaaa gggaacaaaa gctggagctc ttgcatggag gcgcaattgg
      301 caaagacgta aagctcttgt catcaattag atctaatttg gaaacagatt ggtacaattt
      361 ggatggaatt ttaggcttgg cacctatcaa ttgtgagcga cgctttctta cattcaataa
      421 ctgttggttc tgattttcac tttgactatt gccttggtat ggagaatttc tctgcaatgg
      481 ttttgcctgc ttcgaaggag tgctgttact atcgaagata aattccttct tttcaaattg
      541 ccattgtaac atggagttat tctctatatc ctggacgact ggaggagtca agggcctgga
      601 aagtacggat cctgtagaaa tatcactggc aattatactg agtttattgt tggtgttgct
      661 attcatattt gagctggtat tggacgaaag caaggataat gagtttgtgt ctactgactc
      721 caacacccta ggctggattt gatctttctc catcctttaa ttttaacctt ttaattagtg
      781 gttggatcaa gttttcgaga ctattccaat ctgtgacttg ttggataaat agtttttgac
      841 tcgtttagta taatcctttt ttctaaaagt gcttagagtt ctctaagatg ttcttgttta
      901 caatgtgagc gatttaggaa atttcctaaa aatggccgag gcggcgctag cattctacga
      961 aaggtgagat aacgcttcgt tatcgaaaaa tgtcagggga caggggttat ataagaacga
     1021 aaattgtcat cctgcatttt ttctttaaaa cagctataca aaaagtgata ccgacataca
     1081 atgaagtatc taaacttagt tttcgtgctt cagcttctta ttagcatcaa atacgcctca
     1141 ttcggccgag ccttttctct ttttgaagat gataccacct ttgccaattt ggataaacag
     1201 ctaaagcttc cacagaatac acagcaaacc cttaaattgg accgtttgaa tcacgatgat
     1261 ccgctgttta caacttttat ttcttctgtg gacacagatt acagtttgag acttagaaca
     1321 gtagatcctt ctaaactagg aattgacacc gtaaaacaat ggtcgggtta catggactat
     1381 aaggattcca aacacttttt ttactggttt tttgaaagta ggaacgatcc tgctaacgac
     1441 ccaattattc tttggttaaa tggtggacct ggttgttcct cgtttactgg gttgctattt
     1501 gaactaggcc cctcatcaat tggcgccgat atgaaaccaa tccacaatcc ctattcttgg
     1561 aataataacg cttcaatgat cttcttagaa cagccactcg gagtcggctt ttcctatggt
     1621 gatgaaaaag tctcctctac aaaattagca ggcaaagatg cgtacatttt cctggaattg
     1681 ttttttgaag cttttcctca tttacgctcc aacgatttcc acattgcagg cgaagcctat
     1741 gcaggacatt atatccctca aattgcacat gagatcgttg tcaagaaccc tgaaagaacg
     1801 ttcaatttaa cttcagttat gattggtaat ggtatcacag accctttgat tcaagcagat
     1861 tattatgaac caatggcatg cgggaaaggg ggctatcacc ctgttctctc atcagaagaa
     1921 tgtgagaaaa tgagtaaagc tgcaggtcgt tgtcgtaggt tgaacaagtt atgttatgct
     1981 tctaaatcaa gtttaccatg catagtcgcc actgcttact gtgactctgc acttttggaa
     2041 ccgtacatta acacaggact caacgtctat gacattagag ggccctgtga agataatagt
     2101 actgatggta tgtgttatac aggtctccgc tatgtcgacc agtatatgaa ttttcctgaa
     2161 gttcaagaaa cgctagggtc cgacgtgcat aattattctg gctgtgataa tgacgtgttc
     2221 accggatttt tgtttacggg cgatggaagt aaaccatttc aacaatatat tgctgaatta
     2281 ttaaatcaca acattccggt attaatatat gcgggtgata aggcttatat ttgtaattgg
     2341 ctgggaaacc atgcttggtc caatgagttg gaatggatca ataaacgtag gtatcagaga
     2401 aggatgttaa gaccatgggt cagtaaagaa acaggtgaag agttgggaca agtcaagaac
     2461 tatggtcctt tcaccttttt gagaatatac gatgccggtg ctatggtgcc ctatgatcaa
     2521 ccggaggcaa gtttggaaat ggtcaacagt tggatttccg gtaatcgtgc tttttcggat
     2581 ctttccacct tggaaaatgc tagttctagg cggatccccg ggctgcagat gaaaggagaa
     2641 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
     2701 aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa
     2761 tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcact
     2821 tatggtgttc aatgcttttc aagataccca gatcatatga aacggcatga ctttttcaag
     2881 agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac
     2941 tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
     3001 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
     3061 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc
     3121 aaaattagac acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat
     3181 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct
     3241 gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca
     3301 gctgctggga ttacacatgg catggatgaa ctatacaaat agggcgcgcc acttctaaat
     3361 aagcgaattt cttatgattt atgattttta ttattaaata agttataaaa aaaataagtg
     3421 tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa attcttattc ttgagtaact
     3481 ctttcctgta ggtcaggttg ctttctcagg tatagtatga ggtcgctctt attgaccaca
     3541 cctctaccgg gaattcgata tcaagcttat cgataccgtc gacctcgagg gggggcccgg
     3601 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     3661 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     3721 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     3781 gcctgaatgg cgaatggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3841 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3901 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3961 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     4021 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     4081 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     4141 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     4201 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttcctgatg
     4261 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagggta ataactgata
     4321 taattaaatt gaagctctaa tttgtgagtt tagtatacat gcatttactt ataatacagt
     4381 tttttagttt tgctggccgc atcttctcaa atatgcttcc cagcctgctt ttctgtaacg
     4441 ttcaccctct accttagcat cccttccctt tgcaaatagt cctcttccaa caataataat
     4501 gtcagatcct gtagagacca catcatccac ggttctatac tgttgaccca atgcgtctcc
     4561 cttgtcatct aaacccacac cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc
     4621 acccatgtct ctttgagcaa taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc
     4681 aacagtaccc ttagtatatt ctccagtaga tagggagccc ttgcatgaca attctgctaa
     4741 catcaaaagg cctctaggtt cctttgttac ttcttctgcc gcctgcttca aaccgctaac
     4801 aatacctggg cccaccacac cgtgtgcatt cgtaatgtct gcccattctg ctattctgta
     4861 tacacccgca gagtactgca atttgactgt attaccaatg tcagcaaatt ttctgtcttc
     4921 gaagagtaaa aaattgtact tggcggataa tgcctttagc ggcttaactg tgccctccat
     4981 ggaaaaatca gtcaagatat ccacatgtgt ttttagtaaa caaattttgg gacctaatgc
     5041 ttcaactaac tccagtaatt ccttggtggt acgaacatcc aatgaagcac acaagtttgt
     5101 ttgcttttcg tgcatgatat taaatagctt ggcagcaaca ggactaggat gagtagcagc
     5161 acgttcctta tatgtagctt tcgacatgat ttatcttcgt ttcctgcagg tttttgttct
     5221 gtgcagttgg gttaagaata ctgggcaatt tcatgtttct tcaacactac atatgcgtat
     5281 atataccaat ctaagtctgt gctccttcct tcgttcttcc ttctgttcgg agattaccga
     5341 atcaaaaaaa tttcaaggaa accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt
     5401 gaaaaggtgg tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     5461 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5521 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5581 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     5641 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     5701 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5761 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5821 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5881 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5941 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6001 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6061 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6121 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6181 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6241 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6301 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6361 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6421 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6481 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6541 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6601 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6661 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6721 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6781 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6841 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6901 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6961 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7021 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7081 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7141 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7201 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7261 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7321 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7381 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7441 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7501 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7561 accgagcgca gcgagtcagt gagcgaggaa gcgga
//
