LOCUS       sequence-204898-        7667 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Prc1 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 7667)
  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 7667)
  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: pKP113 (pRS406-PRC1p-PRC1-GFP-ADH1t)
FEATURES             Location/Qualifiers
     source          1..7667
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1793..2503
                     /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(1820..1848)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2443..2463
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2527..2714
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2582..2606)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2735..2751)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2736..2752)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2776..2795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2777..2795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2805..2822)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2805..2821)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2814..2836)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2962..3417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3049..3068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3259..3280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3548..4351)
                     /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(4352..4567)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(4591..4610)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4710..4732
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4770..4788)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4856..4960
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4961..5029,5030..5821)
                     /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(5179..5198)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5992..6580
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6481..6500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6734..6751
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6868..6889
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6904..6909,6910..6927,6928..6934)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6942..6958
                     /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     6947..6969
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6966..6982
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6966..6982
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7001..7021
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7003..7021
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tctcaaagaa ggggcccact aataaaagca cgagataaga atgccagcaa aaaagctccg
       61 aaataattct tttcgtcttc cctcctagtc ttaacaagac aagagagaga gagagaaaga
      121 atactcacta gagattgttt cttttctact caacttaaag tatacatacg ctatgaaagc
      181 attcaccagt ttactatgtg gactaggcct gtccactaca ctcgctaagg ccatctcatt
      241 gcaaagaccg ttgggtctag ataaggacgt tttgctgcaa gctgcggaaa aatttggttt
      301 ggacctcgac ctggatcatc tcttgaagga gttggactcc aatgtattgg acgcttgggc
      361 ccaaatagag catttgtacc caaaccaggt tatgagcctt gaaacttcca ctaagccaaa
      421 attccctgaa gcaatcaaaa cgaagaaaga ctgggacttt gtggtcaaga atgacgcaat
      481 tgaaaactat cagcttcgtg tcaacaagat taaggaccct aaaatcctgg gcattgaccc
      541 aaatgtcaca cagtacacgg gttacttgga tgtggaagac gaggacaagc atttcttctt
      601 ttggactttt gaaagtagaa acgatcctgc aaaggatccg gtcatccttt ggttgaacgg
      661 gggtccaggt tgttcttcac taaccgggct gttctttgaa ttaggaccct catccattgg
      721 acctgatttg aaacccatcg ggaaccctta ctcttggaac agcaatgcca ccgtgatctt
      781 ccttgaccag cctgtcaacg ttgggttctc gtattccggg tcctcaggtg tttccaacac
      841 tgtcgccgct ggtaaggatg tctataactt cttggagttg ttcttcgatc agttccctga
      901 atacgtcaac aagggccaag atttccacat cgctggggaa tcctacgccg gccattacat
      961 ccctgttttt gcctctgaaa ttttgtctca caaggacaga aacttcaact taacctccgt
     1021 cttgatcgga aatggcctca ctgacccatt gactcagtat aactattacg aaccaatggc
     1081 ctgtggtgaa ggtggcgaac catctgtttt gccctcggag gaatgctctg ctatggaaga
     1141 ctctttggaa cgttgtttgg gcttgatcga gtcgtgctat gactcgcaat ccgtctggtc
     1201 ctgtgttcca gctaccattt attgtaataa cgcccaattg gctccttacc aacgtaccgg
     1261 cagaaacgtt tacgatatca ggaaggattg tgaaggtggc aatttgtgct acccaacgtt
     1321 acaagatatc gacgactact taaaccagga ctacgtcaaa gaagctgtcg gtgcggaggt
     1381 tgaccactac gaatcctgta acttcgatat caacagaaat ttcctgtttg cgggtgattg
     1441 gatgaagcct taccacaccg ccgtaacaga tcttttgaat caagacctac ccattctggt
     1501 atatgcaggc gataaagatt tcatctgtaa ctggttgggt aataaggcgt ggacggatgt
     1561 cttgccatgg aagtacgacg aagaatttgc aagccaaaaa gtacgtaact ggactgcttc
     1621 tatcaccgac gaggtcgctg gtgaagtcaa atcctacaag cacttcacct atttgagagt
     1681 cttcaatggt ggccacatgg ttccatttga cgtccctgaa aacgccttaa gtatggttaa
     1741 cgaatggatc cacggtggtt tctccttatc tagacggatc cccgggctgc agatgaaagg
     1801 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     1861 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
     1921 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt
     1981 cacttatggt gttcaatgct tttcaagata cccagatcat atgaaacggc atgacttttt
     2041 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
     2101 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
     2161 gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa
     2221 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa
     2281 cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca
     2341 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
     2401 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
     2461 aacagctgct gggattacac atggcatgga tgaactatac aaatagggcg cgccacttct
     2521 aaataagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     2581 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt
     2641 aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc tcttattgac
     2701 cacacctcta ccgggaattc gatatcaagc ttatcgatac cgtcgacctc gagggggggc
     2761 ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     2821 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2881 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2941 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3001 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3061 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3121 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3181 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3241 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3301 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3361 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcct
     3421 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatag ggtaataact
     3481 gatataatta aattgaagct ctaatttgtg agtttagtat acatgcattt acttataata
     3541 cagtttttta gttttgctgg ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt
     3601 aacgttcacc ctctacctta gcatcccttc cctttgcaaa tagtcctctt ccaacaataa
     3661 taatgtcaga tcctgtagag accacatcat ccacggttct atactgttga cccaatgcgt
     3721 ctcccttgtc atctaaaccc acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc
     3781 ttccacccat gtctctttga gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa
     3841 tgtcaacagt acccttagta tattctccag tagataggga gcccttgcat gacaattctg
     3901 ctaacatcaa aaggcctcta ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc
     3961 taacaatacc tgggcccacc acaccgtgtg cattcgtaat gtctgcccat tctgctattc
     4021 tgtatacacc cgcagagtac tgcaatttga ctgtattacc aatgtcagca aattttctgt
     4081 cttcgaagag taaaaaattg tacttggcgg ataatgcctt tagcggctta actgtgccct
     4141 ccatggaaaa atcagtcaag atatccacat gtgtttttag taaacaaatt ttgggaccta
     4201 atgcttcaac taactccagt aattccttgg tggtacgaac atccaatgaa gcacacaagt
     4261 ttgtttgctt ttcgtgcatg atattaaata gcttggcagc aacaggacta ggatgagtag
     4321 cagcacgttc cttatatgta gctttcgaca tgatttatct tcgtttcctg caggtttttg
     4381 ttctgtgcag ttgggttaag aatactgggc aatttcatgt ttcttcaaca ctacatatgc
     4441 gtatatatac caatctaagt ctgtgctcct tccttcgttc ttccttctgt tcggagatta
     4501 ccgaatcaaa aaaatttcaa ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg
     4561 aattgaaaag gtggtatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4621 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4681 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4741 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4801 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4861 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4921 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4981 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5041 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5101 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5161 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5221 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5281 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5341 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5401 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5461 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5521 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5581 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5641 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5701 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5761 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5821 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5881 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5941 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6001 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6061 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6121 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6181 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6241 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6301 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6361 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6421 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6481 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6541 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6601 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6661 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6721 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     6781 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     6841 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     6901 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6961 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
     7021 ggaacaaaag ctggagctcg aatgagtgaa aaatttgtta gcagagaagc aagaaggaaa
     7081 gaaggaaaaa aaaaagaaac cgtgtaagaa atcatggcgc ctcaaaaaaa aaaaaatacg
     7141 cgaaatcgat ttccgtatat gatgatacat atgttaggtc tcttatggta gtttttaggg
     7201 tctgtcctgt tttttgaagg cattggttag ggtctagtaa gtaactcgta taaagagatg
     7261 tacttttata cggtatgtgt ggcggttatt tccagtgttt tttttccctt attttgtggg
     7321 ttcatggaaa tagtacatat tgaccttctg cacaagaagc catattgaca gagcagtatg
     7381 tgaggacctt tcttctacac aatgtaacag tcaatggttg tagtcaatac cacgaccttt
     7441 acgtgcagtt ttagagcgaa acttcggttt tttgagactt acctcgtgtt tgtgtctccc
     7501 tgggaatgga gccgcgccac ccttgtctat tatgtatagt cttatttaat catagcgatg
     7561 ttggtcatcc agtacacttc ggtagcaacc ttcgtttgtg attgtcttgg taattgcttc
     7621 caacaacttt atccatcatt gagacagggg ccatatcacc cgcgggg
//
