LOCUS       sequence-201584-        5306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for creating C-terminal GFP fusion proteins in yeast..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5306)
  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 5306)
  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: pKP112 (pRS406-GFP-ADH1t)
FEATURES             Location/Qualifiers
     source          1..5306
                     /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"
     primer_bind     2298..2314
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2298..2314
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2328..3038
                     /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(2355..2383)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2978..2998
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3062..3249
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3117..3141)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3270..3286)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3271..3287)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3311..3330)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3312..3330)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3340..3357)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3340..3356)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3349..3371)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3501..3956
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3588..3607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3798..3819
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4087..4890)
                     /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(4891..5111)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5137..5156)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5256..5278
                     /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 ctggagctcc
     2281 accgcggtgg cggccgctct agaactagtg gatcccccgg gctgcagatg aaaggagaag
     2341 aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca
     2401 aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat
     2461 ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact actttcactt
     2521 atggtgttca atgcttttca agatacccag atcatatgaa acggcatgac tttttcaaga
     2581 gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat gacgggaact
     2641 acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga atcgagttaa
     2701 aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa tacaactata
     2761 actcacacaa tgtatacatc atggcagaca aacaaaagaa tggaatcaaa gttaacttca
     2821 aaattagaca caacattgaa gatggaagcg ttcaactagc agaccattat caacaaaata
     2881 ctccaattgg cgatggccct gtccttttac cagacaacca ttacctgtcc acacaatctg
     2941 ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag
     3001 ctgctgggat tacacatggc atggatgaac tatacaaata gggcgcgcca cttctaaata
     3061 agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt
     3121 atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc
     3181 tttcctgtag gtcaggttgc tttctcaggt atagtatgag gtcgctctta ttgaccacac
     3241 ctctaccggg aattcgatat caagcttatc gataccgtcg acctcgaggg ggggcccggt
     3301 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     3361 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3421 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3481 cctgaatggc gaatggcgcg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3541 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3601 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3661 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3721 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3781 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3841 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3901 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttcctg
     3961 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatagg gtaataactg
     4021 atataattaa attgaagctc taatttgtga gtttagtata catgcattta cttataatac
     4081 agttttttag ttttgctggc cgcatcttct caaatatgct tcccagcctg cttttctgta
     4141 acgttcaccc tctaccttag catcccttcc ctttgcaaat agtcctcttc caacaataat
     4201 aatgtcagat cctgtagaga ccacatcatc cacggttcta tactgttgac ccaatgcgtc
     4261 tcccttgtca tctaaaccca caccgggtgt cataatcaac caatcgtaac cttcatctct
     4321 tccacccatg tctctttgag caataaagcc gataacaaaa tctttgtcgc tcttcgcaat
     4381 gtcaacagta cccttagtat attctccagt agatagggag cccttgcatg acaattctgc
     4441 taacatcaaa aggcctctag gttcctttgt tacttcttct gccgcctgct tcaaaccgct
     4501 aacaatacct gggcccacca caccgtgtgc attcgtaatg tctgcccatt ctgctattct
     4561 gtatacaccc gcagagtact gcaatttgac tgtattacca atgtcagcaa attttctgtc
     4621 ttcgaagagt aaaaaattgt acttggcgga taatgccttt agcggcttaa ctgtgccctc
     4681 catggaaaaa tcagtcaaga tatccacatg tgtttttagt aaacaaattt tgggacctaa
     4741 tgcttcaact aactccagta attccttggt ggtacgaaca tccaatgaag cacacaagtt
     4801 tgtttgcttt tcgtgcatga tattaaatag cttggcagca acaggactag gatgagtagc
     4861 agcacgttcc ttatatgtag ctttcgacat gatttatctt cgtttcctgc aggtttttgt
     4921 tctgtgcagt tgggttaaga atactgggca atttcatgtt tcttcaacac tacatatgcg
     4981 tatatatacc aatctaagtc tgtgctcctt ccttcgttct tccttctgtt cggagattac
     5041 cgaatcaaaa aaatttcaaa gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga
     5101 attgaattga aaagctgtgg tatggtgcac tctcagtaca atctgctctg atgccgcata
     5161 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5221 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5281 ttcaccgtca tcaccgaaac gcgcga
//
