LOCUS       sequence-204896-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Atg42/Ybr139w(S219A) 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: pKP129 [pRS406-YBR139Wp-YBR139W(S219A)-GFP-ADH1t]
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    4..25
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(40..45,46..63,64..70)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    78..94
                     /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     83..105
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     102..118
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     102..118
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     137..157
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        139..157
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2524..3234
                     /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(2551..2579)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3174..3194
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3258..3445
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3313..3337)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3466..3482)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3467..3483)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3507..3526)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3508..3526)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3536..3553)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3536..3552)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3545..3567)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3693..4148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3780..3799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3990..4011
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4279..5082)
                     /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(5083..5298)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5322..5341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5441..5463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5501..5519)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5587..5691
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5692..5760,5761..6552)
                     /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(5910..5929)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6723..7311
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7212..7231
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7465..7482
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
       61 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
      121 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctcttgca
      181 tggaggcgca attggcaaag acgtaaagct cttgtcatca attagatcta atttggaaac
      241 agattggtac aatttggatg gaattttagg cttggcacct atcaattgtg agcgacgctt
      301 tcttacattc aataactgtt ggttctgatt ttcactttga ctattgcctt ggtatggaga
      361 atttctctgc aatggttttg cctgcttcga aggagtgctg ttactatcga agataaattc
      421 cttcttttca aattgccatt gtaacatgga gttattctct atatcctgga cgactggagg
      481 agtcaagggc ctggaaagta cggatcctgt agaaatatca ctggcaatta tactgagttt
      541 attgttggtg ttgctattca tatttgagct ggtattggac gaaagcaagg ataatgagtt
      601 tgtgtctact gactccaaca ccctaggctg gatttgatct ttctccatcc tttaatttta
      661 accttttaat tagtggttgg atcaagtttt cgagactatt ccaatctgtg acttgttgga
      721 taaatagttt ttgactcgtt tagtataatc cttttttcta aaagtgctta gagttctcta
      781 agatgttctt gtttacaatg tgagcgattt aggaaatttc ctaaaaatgg ccgaggcggc
      841 gctagcattc tacgaaaggt gagataacgc ttcgttatcg aaaaatgtca ggggacaggg
      901 gttatataag aacgaaaatt gtcatcctgc attttttctt taaaacagct atacaaaaag
      961 tgataccgac atacaatgaa gtatctaaac ttagttttcg tgcttcagct tcttattagc
     1021 atcaaatacg cctcattcgg ccgagccttt tctctttttg aagatgatac cacctttgcc
     1081 aatttggata aacagctaaa gcttccacag aatacacagc aaacccttaa attggaccgt
     1141 ttgaatcacg atgatccgct gtttacaact tttatttctt ctgtggacac agattacagt
     1201 ttgagactta gaacagtaga tccttctaaa ctaggaattg acaccgtaaa acaatggtcg
     1261 ggttacatgg actataagga ttccaaacac tttttttact ggttttttga aagtaggaac
     1321 gatcctgcta acgacccaat tattctttgg ttaaatggtg gacctggttg ttcctcgttt
     1381 actgggttgc tatttgaact aggcccctca tcaattggcg ccgatatgaa accaatccac
     1441 aatccctatt cttggaataa taacgcttca atgatcttct tagaacagcc actcggagtc
     1501 ggcttttcct atggtgatga aaaagtctcc tctacaaaat tagcaggcaa agatgcgtac
     1561 attttcctgg aattgttttt tgaagctttt cctcatttac gctccaacga tttccacatt
     1621 gcaggcgaag cctatgcagg acattatatc cctcaaattg cacatgagat cgttgtcaag
     1681 aaccctgaaa gaacgttcaa tttaacttca gttatgattg gtaatggtat cacagaccct
     1741 ttgattcaag cagattatta tgaaccaatg gcatgcggga aagggggcta tcaccctgtt
     1801 ctctcatcag aagaatgtga gaaaatgagt aaagctgcag gtcgttgtcg taggttgaac
     1861 aagttatgtt atgcttctaa atcaagttta ccatgcatag tcgccactgc ttactgtgac
     1921 tctgcacttt tggaaccgta cattaacaca ggactcaacg tctatgacat tagagggccc
     1981 tgtgaagata atagtactga tggtatgtgt tatacaggtc tccgctatgt cgaccagtat
     2041 atgaattttc ctgaagttca agaaacgcta gggtccgacg tgcataatta ttctggctgt
     2101 gataatgacg tgttcaccgg atttttgttt acgggcgatg gaagtaaacc atttcaacaa
     2161 tatattgctg aattattaaa tcacaacatt ccggtattaa tatatgcggg tgataaggat
     2221 tatatttgta attggctggg aaaccatgct tggtccaatg agttggaatg gatcaataaa
     2281 cgtaggtatc agagaaggat gttaagacca tgggtcagta aagaaacagg tgaagagttg
     2341 ggacaagtca agaactatgg ccctttcacc tttttgagaa tatacgatgc cggtcatatg
     2401 gtgccctatg atcaaccgga ggcaagtttg gaaatggtca acagttggat ttccggtaat
     2461 cgtgcttttt cggatctttc caccttggaa aatgctagtt ctagacggat ccccgggctg
     2521 cagatgaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
     2581 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga
     2641 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt
     2701 gtcactactt tcacttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacgg
     2761 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc
     2821 aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt
     2881 aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa
     2941 ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga
     3001 atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac
     3061 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
     3121 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt
     3181 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaatagggc
     3241 gcgccacttc taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta
     3301 taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct
     3361 tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg
     3421 ctcttattga ccacacctct accgggaatt cgatatcaag cttatcgata ccgtcgacct
     3481 cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg
     3541 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     3601 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     3661 cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc
     3721 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3781 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3841 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3901 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3961 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4021 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4081 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4141 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4201 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt
     4261 tacttataat acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc
     4321 tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct
     4381 tccaacaata ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg
     4441 acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta
     4501 accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc
     4561 gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca
     4621 tgacaattct gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg
     4681 cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca
     4741 ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc
     4801 aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt
     4861 aactgtgccc tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat
     4921 tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga
     4981 agcacacaag tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact
     5041 aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct
     5101 gcaggttttt gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac
     5161 actacatatg cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg
     5221 ttcggagatt accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa
     5281 aaaaaatgat gaattgaaaa ggtggtatgg tgcactctca gtacaatctg ctctgatgcc
     5341 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5401 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5461 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5521 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5581 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5641 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5701 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     5761 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     5821 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     5881 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     5941 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6001 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6061 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6121 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6181 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6241 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6301 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6361 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     6421 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6481 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6541 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6601 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6661 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6721 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     6781 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     6841 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     6901 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     6961 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7021 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7081 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7141 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7201 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7261 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7321 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     7381 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     7441 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     7501 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     7561 tttcccgact ggaaagcggg cagtgagcgc aacgc
//
