LOCUS       sequence-490856-        5172 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  To endogenously tag S. cerevisiae proteins at their C-terminus with 
            Pho8∆60; TRP1 selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5172)
  AUTHORS   Valdbjorn Kanne J, Reggiori F
  TITLE     A plasmid module for PCR-based gene modification for the accurate 
            measurement of vacuolar delivery of specific proteins in yeast 
            Saccharomyces cerevisiae.
  JOURNAL   Autophagy Rep. 2025 May 31;4(1):2511724. doi: 
            10.1080/27694127.2025.2511724. eCollection 2025.
  PUBMED    40458443
REFERENCE   2  (bases 1 to 5172)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Autophagy 
            Rep."; date: "2025-05-31"; volume: "4(1):2511724. doi"; pages: " 
            10.1080/27694127.2025.2511724. eCollection 2025"
COMMENT     Sequence Label: pFA6a-Pho8d∆60-TRP1
FEATURES             Location/Qualifiers
     source          1..5172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1602..1789
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1657..1681)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        1825..1972
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             1973..2647
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     RBS             2727..2735
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(2807..2826)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2808..2826)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2913..2930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3084..3672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3164..3183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3843..4634,4635..4703))
                     /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     4466..4485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4704..4808)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4876..4894
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4932..4954)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5054..5073
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5154..5172
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5154..5171
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 acgcggccgc cagctgaagc ttcgtacgct gcaggtcgac ggatccccgg gttaattaag
       61 tctgcatcac acaagaagaa gaatgtcata ttcttcgtga cggatgggat gggacctgcg
      121 tctttgtcca tggcaagatc attcaaccag catgttaatg atttacctat agacgacatc
      181 ctgacacttg acgagcattt tatcgggtct tcgagaacaa ggtcctcgga ctcactagtc
      241 actgactcag ctgcgggcgc caccgcattt gcatgtgcat taaagagcta taatggggcc
      301 atcggtgttg atccgcatca cagaccttgt ggtacagtgc tcgaggcggc taagttagca
      361 ggttatctca ctgggcttgt tgttacgaca agaatcacag atgccactcc ggcttcattc
      421 agttcacatg ttgattacag atggcaggaa gatcttatcg ctacgcacca gttaggcgaa
      481 tatcctctgg ggagagtagt agacttactc atgggtggcg gcaggagcca tttctaccct
      541 caaggcgaaa aggcttctcc atatggtcac cacggtgcta gaaaagatgg aagagattta
      601 atcgatgagg ctcaatctaa tggctggcag tacgtgggtg accgtaaaaa ttttgattct
      661 ttgttgaaaa gccacggtga aaacgttact ttacctttct tgggtctctt tgcagataac
      721 gacattcctt ttgaaattga cagagatgaa aaggagtatc cttcccttaa ggaacaagtc
      781 aaggtggctt tgggtgcctt ggaaaaagct tccaatgagg acaaagattc taatgggttt
      841 ttcttaatgg tggaaggctc cagaattgat catgctggtc accaaaacga tccagcttcg
      901 caggtaaggg aggtgttggc tttcgatgaa gcattccaat acgttttgga gtttgctgaa
      961 aattccgata cggagactgt tttggtctct acgtcggatc atgaaacggg tggtttagtc
     1021 acttcaagac aagtcactgc tagctatcct cagtatgtct ggtatccaca ggtgctcgcc
     1081 aacgccaccc attcaggtga gtttttgaaa agaaaactag tagattttgt tcatgagcat
     1141 aagggcgcaa gcagcaaaat agaaaatttc atcaaacatg agatcctgga aaaagatttg
     1201 ggtatttacg attacacgga tagcgattta gaaacattaa ttcatttgga tgacaacgca
     1261 aatgctatcc aagacaaact gaatgacatg gtatcattta gggcccaaat tggctggacc
     1321 acgcatggtc acagcgcagt cgacgtcaac atatatgcat acgccaataa gaaagctaca
     1381 tggtcatatg tcttgaacaa tttacaaggc aatcacgaaa atacggaagt tggccagttc
     1441 ttagaaaact ttttagaatt gaacctgaac gaagtcactg atttgattag ggacaccaaa
     1501 cacacatcag attttgatgc tacagaaata gctagtgaag tacaacatta tgacgaatac
     1561 taccatgcgt tgaccaactg aggcgcgcca cttctaaata agcgaatttc ttatgattta
     1621 tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt taaagtgact
     1681 cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag gtcaggttgc
     1741 tttctcaggt atagtatgag gtcgctctta ttgaccacac ctctaccggc agatccgcta
     1801 gggataacag ggtaatatag atctgtacaa tcttgatccg gagcttttct ttttttgccg
     1861 attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag ggcattggtg
     1921 actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga gtatgtctgt
     1981 tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc agagcacaga
     2041 ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat gtgtgcccaa
     2101 tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg taaaagcata
     2161 taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc aacctaagga
     2221 ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc atggagatga
     2281 gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac tcgtatttcc
     2341 aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt ttattccctt
     2401 gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga tttctgactg
     2461 ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg gactgacgcc
     2521 agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg taagcggagg
     2581 tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca aaaatgctaa
     2641 gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt gccgatctat
     2701 gcggtgtgaa ataccgcaca gatgcgtaag gaggtttaaa cgagctcgaa ttcatcgatg
     2761 atatcagatc cactagtggc ctatgcggcc gcggatctgc cggtctccct atagtgagtc
     2821 gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga
     2881 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2941 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3001 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3061 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3121 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3181 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3241 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3301 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3361 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3421 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3481 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3541 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3601 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3661 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3721 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3781 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3841 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3901 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     3961 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4021 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4081 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4141 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4201 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4261 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4321 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4381 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4441 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4501 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4561 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4621 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     4681 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     4741 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     4801 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     4861 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     4921 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     4981 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     5041 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
     5101 tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg
     5161 tgacactata ga
//
