LOCUS       sequence-204908-        5295 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 5295)
  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 5295)
  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..5295
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    120..141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(156..161,162..179,180..186)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    194..210
                     /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     199..221
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     218..234
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     218..234
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     253..273
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        255..273
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     310..326
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     310..326
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             340..1050
                     /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(367..395)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     990..1010
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1074..1261
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1129..1153)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(1282..1298)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1283..1299)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(1323..1342)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1324..1342)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1352..1369)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1352..1368)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1361..1383)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1509..1964
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1596..1615)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1806..1827
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2095..2898)
                     /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(2899..3114)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3138..3157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3257..3279
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3317..3335)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3403..3507
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3508..3576,3577..4368)
                     /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(3726..3745)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4539..5127
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5028..5047
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(5281..5295,1..3)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
       61 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
      121 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
      181 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
      241 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt
      301 ggcggccgct ctagaactag tggatccccc gggctgcaga tgaaaggaga agaacttttc
      361 actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct
      421 gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc
      481 actactggaa aactacctgt tccatggcca acacttgtca ctactttcac ttatggtgtt
      541 caatgctttt caagataccc agatcatatg aaacggcatg actttttcaa gagtgccatg
      601 cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca
      661 cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt
      721 gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac
      781 aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga
      841 cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt
      901 ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg
      961 aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg
     1021 attacacatg gcatggatga actatacaaa tagggcgcgc cacttctaaa taagcgaatt
     1081 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
     1141 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
     1201 aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg
     1261 ggaattcgat atcaagctta tcgataccgt cgacctcgag ggggggcccg gtacccaatt
     1321 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     1381 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     1441 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     1501 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1561 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1621 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1681 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1741 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1801 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1861 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1921 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt
     1981 ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat
     2041 tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt
     2101 ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc
     2161 taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc
     2221 tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc
     2281 taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc
     2341 tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc
     2401 cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag
     2461 gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg
     2521 gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc
     2581 agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa
     2641 aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc
     2701 agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa
     2761 ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc
     2821 gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt
     2881 atatgtagct ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg
     2941 ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa
     3001 tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa
     3061 atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaaggtg
     3121 gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     3181 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3241 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3301 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3361 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3421 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3481 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3541 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3601 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3661 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3721 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     3781 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3841 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3901 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3961 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4021 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4081 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4141 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4201 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4261 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4321 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4381 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4441 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4501 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4561 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4621 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4681 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4741 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4801 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4861 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4921 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4981 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5041 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5101 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5161 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5221 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5281 agcgagtcag tgagc
//
