LOCUS       sequence-204902-        7595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Atg42/Ybr139w(D415A) 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: pKP133 [pRS406-YBR139Wp-YBR139W(D415A)-GFP-ADH1t]
FEATURES             Location/Qualifiers
     source          1..7595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(129..134,135..152,153..159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    167..183
                     /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     172..194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     191..207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     191..207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     226..246
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        228..246
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2613..3323
                     /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(2640..2668)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3263..3283
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3347..3534
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3402..3426)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3555..3571)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3556..3572)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3596..3615)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3597..3615)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3625..3642)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3625..3641)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3634..3656)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3782..4237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3869..3888)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4079..4100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4368..5171)
                     /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(5172..5387)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5411..5430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5530..5552
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5590..5608)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5676..5780
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5781..5849,5850..6641)
                     /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(5999..6018)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6812..7400
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7301..7320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7554..7571
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      241 taaagggaac aaaagctgga gctcttgcat ggaggcgcaa ttggcaaaga cgtaaagctc
      301 ttgtcatcaa ttagatctaa tttggaaaca gattggtaca atttggatgg aattttaggc
      361 ttggcaccta tcaattgtga gcgacgcttt cttacattca ataactgttg gttctgattt
      421 tcactttgac tattgccttg gtatggagaa tttctctgca atggttttgc ctgcttcgaa
      481 ggagtgctgt tactatcgaa gataaattcc ttcttttcaa attgccattg taacatggag
      541 ttattctcta tatcctggac gactggagga gtcaagggcc tggaaagtac ggatcctgta
      601 gaaatatcac tggcaattat actgagttta ttgttggtgt tgctattcat atttgagctg
      661 gtattggacg aaagcaagga taatgagttt gtgtctactg actccaacac cctaggctgg
      721 atttgatctt tctccatcct ttaattttaa ccttttaatt agtggttgga tcaagttttc
      781 gagactattc caatctgtga cttgttggat aaatagtttt tgactcgttt agtataatcc
      841 ttttttctaa aagtgcttag agttctctaa gatgttcttg tttacaatgt gagcgattta
      901 ggaaatttcc taaaaatggc cgaggcggcg ctagcattct acgaaaggtg agataacgct
      961 tcgttatcga aaaatgtcag gggacagggg ttatataaga acgaaaattg tcatcctgca
     1021 ttttttcttt aaaacagcta tacaaaaagt gataccgaca tacaatgaag tatctaaact
     1081 tagttttcgt gcttcagctt cttattagca tcaaatacgc ctcattcggc cgagcctttt
     1141 ctctttttga agatgatacc acctttgcca atttggataa acagctaaag cttccacaga
     1201 atacacagca aacccttaaa ttggaccgtt tgaatcacga tgatccgctg tttacaactt
     1261 ttatttcttc tgtggacaca gattacagtt tgagacttag aacagtagat ccttctaaac
     1321 taggaattga caccgtaaaa caatggtcgg gttacatgga ctataaggat tccaaacact
     1381 ttttttactg gttttttgaa agtaggaacg atcctgctaa cgacccaatt attctttggt
     1441 taaatggtgg acctggttgt tcctcgttta ctgggttgct atttgaacta ggcccctcat
     1501 caattggcgc cgatatgaaa ccaatccaca atccctattc ttggaataat aacgcttcaa
     1561 tgatcttctt agaacagcca ctcggagtcg gcttttccta tggtgatgaa aaagtctcct
     1621 ctacaaaatt agcaggcaaa gatgcgtaca ttttcctgga attgtttttt gaagcttttc
     1681 ctcatttacg ctccaacgat ttccacattg caggcgaatc ctatgcagga cattatatcc
     1741 ctcaaattgc acatgagatc gttgtcaaga accctgaaag aacgttcaat ttaacttcag
     1801 ttatgattgg taatggtatc acagaccctt tgattcaagc agattattat gaaccaatgg
     1861 catgcgggaa agggggctat caccctgttc tctcatcaga agaatgtgag aaaatgagta
     1921 aagctgcagg tcgttgtcgt aggttgaaca agttatgtta tgcttctaaa tcaagtttac
     1981 catgcatagt cgccactgct tactgtgact ctgcactttt ggaaccgtac attaacacag
     2041 gactcaacgt ctatgacatt agagggccct gtgaagataa tagtactgat ggtatgtgtt
     2101 atacaggtct ccgctatgtc gaccagtata tgaattttcc tgaagttcaa gaaacgctag
     2161 ggtccgacgt gcataattat tctggctgtg ataatgacgt gttcaccgga tttttgttta
     2221 cgggcgatgg aagtaaacca tttcaacaat atattgctga attattaaat cacaacattc
     2281 cggtattaat atatgcgggt gataaggctt atatttgtaa ttggctggga aaccatgctt
     2341 ggtccaatga gttggaatgg atcaataaac gtaggtatca gagaaggatg ttaagaccat
     2401 gggtcagtaa agaaacaggt gaagagttgg gacaagtcaa gaactatggc cctttcacct
     2461 ttttgagaat atacgatgcc ggtcatatgg tgccctatga tcaaccggag gcaagtttgg
     2521 aaatggtcaa cagttggatt tccggtaatc gtgctttttc ggatctttcc accttggaaa
     2581 atgctagttc tagacggatc cccgggctgc agatgaaagg agaagaactt ttcactggag
     2641 ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg
     2701 gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg
     2761 gaaaactacc tgttccatgg ccaacacttg tcactacttt cacttatggt gttcaatgct
     2821 tttcaagata cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag
     2881 gttatgtaca ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg
     2941 aagtcaagtt tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta
     3001 aagaagatgg aaacattctt ggacacaaat tggaatacaa ctataactca cacaatgtat
     3061 acatcatggc agacaaacaa aagaatggaa tcaaagttaa cttcaaaatt agacacaaca
     3121 ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg
     3181 gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc
     3241 ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac
     3301 atggcatgga tgaactatac aaatagggcg cgccacttct aaataagcga atttcttatg
     3361 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
     3421 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
     3481 gttgctttct caggtatagt atgaggtcgc tcttattgac cacacctcta ccgggaattc
     3541 gatatcaagc ttatcgatac cgtcgacctc gagggggggc ccggtaccca attcgcccta
     3601 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3661 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3721 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3781 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3841 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3901 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3961 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4021 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4081 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4141 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4201 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     4261 cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta aattgaagct
     4321 ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta gttttgctgg
     4381 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     4441 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     4501 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     4561 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     4621 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     4681 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     4741 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     4801 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     4861 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     4921 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     4981 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     5041 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     5101 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     5161 gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag
     5221 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     5281 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     5341 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag gtggtatggt
     5401 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5461 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5521 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5581 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5641 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5701 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5761 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5821 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5881 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5941 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6001 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6061 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6121 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6181 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6241 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6301 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6361 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6421 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6481 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6541 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6601 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6661 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6721 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6781 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6841 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6901 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6961 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7021 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7081 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7141 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7201 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7261 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7321 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7381 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7441 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7501 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7561 cagtgagcga ggaagcggaa gagcgcccaa tacgc
//
