LOCUS       sequence-201586-        7678 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 7678)
  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 7678)
  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..7678
                     /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             4700..5410
                     /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(4727..4755)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5350..5370
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      5434..5621
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5489..5513)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5642..5658)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5643..5659)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(5683..5702)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5684..5702)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5712..5729)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5712..5728)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5721..5743)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5873..6328
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5960..5979)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6170..6191
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6459..7262)
                     /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(7263..7483)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7509..7528)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7628..7650
                     /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 ctggagctcg
     2281 aatgagtgaa aaatttgtta gcagagaagc aagaaggaaa gaaggaaaaa aaaaagaaac
     2341 cgtgtaagaa atcatggcgc ctcaaaaaaa aaaaaatacg cgaaatcgat ttccgtatat
     2401 gatgatacat atgttaggtc tcttatggta gtttttaggg tctgtcctgt tttttgaagg
     2461 cattggttag ggtctagtaa gtaactcgta taaagagatg tacttttata cggtatgtgt
     2521 ggcggttatt tccagtgttt tttttccctt attttgtggg ttcatggaaa tagtacatat
     2581 tgaccttctg cacaagaagc catattgaca gagcagtatg tgaggacctt tcttctacac
     2641 aatgtaacag tcaatggttg tagtcaatac cacgaccttt acgtgcagtt ttagagcgaa
     2701 acttcggttt tttgagactt acctcgtgtt tgtgtctccc tgggaatgga gccgcgccac
     2761 ccttgtctat tatgtatagt cttatttaat catagcgatg ttggtcatcc agtacacttc
     2821 ggtagcaacc ttcgtttgtg attgtcttgg taattgcttc caacaacttt atccatcatt
     2881 gagacagggg ccatatcacc cgcggggtct caaagaaggg gcccactaat aaaagcacga
     2941 gataagaatg ccagcaaaaa agctccgaaa taattctttt cgtcttccct cctagtctta
     3001 acaagacaag agagagagag agaaagaata ctcactagag attgtttctt ttctactcaa
     3061 cttaaagtat acatacgcta tgaaagcatt caccagttta ctatgtggac taggcctgtc
     3121 cactacactc gctaaggcca tctcattgca aagaccgttg ggtctagata aggacgtttt
     3181 gctgcaagct gcggaaaaat ttggtttgga cctcgacctg gatcatctct tgaaggagtt
     3241 ggactccaat gtattggacg cttgggccca aatagagcat ttgtacccaa accaggttat
     3301 gagccttgaa acttccacta agccaaaatt ccctgaagca atcaaaacga agaaagactg
     3361 ggactttgtg gtcaagaatg acgcaattga aaactatcag cttcgtgtca acaagattaa
     3421 ggaccctaaa atcctgggca ttgacccaaa tgtcacacag tacacgggtt acttggatgt
     3481 ggaagacgag gacaagcatt tcttcttttg gacttttgaa agtagaaacg atcctgcaaa
     3541 ggatccggtc atcctttggt tgaacggggg tccaggttgt tcttcactaa ccgggctgtt
     3601 ctttgaatta ggaccctcat ccattggacc tgatttgaaa cccatcggga acccttactc
     3661 ttggaacagc aatgccaccg tgatcttcct tgaccagcct gtcaacgttg ggttctcgta
     3721 ttccgggtcc tcaggtgttt ccaacactgt cgccgctggt aaggatgtct ataacttctt
     3781 ggagttgttc ttcgatcagt tccctgaata cgtcaacaag ggccaagatt tccacatcgc
     3841 tggggaatcc tacgccggcc attacatccc tgtttttgcc tctgaaattt tgtctcacaa
     3901 ggacagaaac ttcaacttaa cctccgtctt gatcggaaat ggcctcactg acccattgac
     3961 tcagtataac tattacgaac caatggcctg tggtgaaggt ggcgaaccat ctgttttgcc
     4021 ctcggaggaa tgctctgcta tggaagactc tttggaacgt tgtttgggct tgatcgagtc
     4081 gtgctatgac tcgcaatccg tctggtcctg tgttccagct accatttatt gtaataacgc
     4141 ccaattggct ccttaccaac gtaccggcag aaacgtttac gatatcagga aggattgtga
     4201 aggtggcaat ttgtgctacc caacgttaca agatatcgac gactacttaa accaggacta
     4261 cgtcaaagaa gctgtcggtg cggaggttga ccactacgaa tcctgtaact tcgatatcaa
     4321 cagaaatttc ctgtttgcgg gtgattggat gaagccttac cacaccgccg taacagatct
     4381 tttgaatcaa gacctaccca ttctggtata tgcaggcgat aaagatttca tctgtaactg
     4441 gttgggtaat aaggcgtgga cggatgtctt gccatggaag tacgacgaag aatttgcaag
     4501 ccaaaaagta cgtaactgga ctgcttctat caccgacgag gtcgctggtg aagtcaaatc
     4561 ctacaagcac ttcacctatt tgagagtctt caatggtggc cacatggttc catttgacgt
     4621 ccctgaaaac gccttaagta tggttaacga atggatccac ggtggtttct ccttatctag
     4681 acggatcccc gggctgcaga tgaaaggaga agaacttttc actggagttg tcccaattct
     4741 tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg
     4801 tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt
     4861 tccatggcca acacttgtca ctactttcac ttatggtgtt caatgctttt caagataccc
     4921 agatcatatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga
     4981 aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag tcaagtttga
     5041 aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa
     5101 cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga
     5161 caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag
     5221 cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt
     5281 accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag
     5341 agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga
     5401 actatacaaa tagggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt
     5461 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     5521 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     5581 gtatagtatg aggtcgctct tattgaccac acctctaccg ggaattcgat atcaagctta
     5641 tcgataccgt cgacctcgag ggggggcccg gtacccaatt cgccctatag tgagtcgtat
     5701 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5761 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     5821 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc
     5881 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5941 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     6001 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     6061 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     6121 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     6181 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     6241 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     6301 tttaacaaaa tattaacgtt tacaatttcc tgatgcggta ttttctcctt acgcatctgt
     6361 gcggtatttc acaccgcata gggtaataac tgatataatt aaattgaagc tctaatttgt
     6421 gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg gccgcatctt
     6481 ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt
     6541 ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga gaccacatca
     6601 tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt
     6661 gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag
     6721 ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt atattctcca
     6781 gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct aggttccttt
     6841 gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt
     6901 gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg
     6961 actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg
     7021 gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa gatatccaca
     7081 tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag taattccttg
     7141 gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat gatattaaat
     7201 agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac
     7261 atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa gaatactggg
     7321 caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag tctgtgctcc
     7381 ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca aagaaaccga
     7441 aatcaaaaaa aagaataaaa aaaaaatgat gaattgaatt gaaaagctgt ggtatggtgc
     7501 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     7561 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     7621 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga
//
