LOCUS       sequence-490854-        5452 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  To endogenously tag S. cerevisiae proteins at their C-terminus with 
            Pho8∆60; NatMX6 selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5452)
  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 5452)
  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-Pho8∆60-NatMX6
FEATURES             Location/Qualifiers
     source          1..5452
                     /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"
     gene            1864..2983
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        1864..2207
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2208..2780
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      2786..2983
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3087..3106)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3088..3106)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3193..3210)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3364..3952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3444..3463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4123..4914,4915..4983))
                     /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     4746..4765
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4984..5088)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5156..5174
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5212..5234)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5334..5353
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5434..5452
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5434..5451
                     /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 atctgtttag cttgcctcgt ccccgccggg tcacccggcc
     1861 agcgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca
     1921 tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc
     1981 atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcagacctgc
     2041 gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag
     2101 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tcctttttaa
     2161 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaccactc
     2221 ttgacgacac ggcttaccgg taccgcacca gtgtcccggg ggacgccgag gccatcgagg
     2281 cactggatgg gtccttcacc accgacaccg tcttccgcgt caccgccacc ggggacggct
     2341 tcaccctgcg ggaggtgccg gtggacccgc ccctgaccaa ggtgttcccc gacgacgaat
     2401 cggacgacga atcggacgac ggggaggacg gcgacccgga ctctcggacg ttcgtcgcgt
     2461 acggggacga cggcgacctg gcgggcttcg tggtcgtctc gtactccggc tggaaccgcc
     2521 ggctgaccgt cgaggacatc gaggtcgccc cggagcaccg ggggcacggg gtcgggcgcg
     2581 cgttgatggg gctcgcgacg gagttcgccc gcgagcgggg tgccgggcac ctctggctgg
     2641 aggtcaccaa cgtcaacgca ccggcgatcc acgcgtaccg gcggatgggg ttcaccctct
     2701 gcggcctgga caccgccctg tacgacggca ccgcctcgga cggcgagcag gcgctctaca
     2761 tgagcatgcc ctgcccctaa tcagtactga caataaaaag attcttgttt tcaagaactt
     2821 gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg ttagcgtgat
     2881 ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg cgcagaaagt
     2941 aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgctgtcga ttcgatacta
     3001 acgccgccat ccagtttaaa cgagctcgaa ttcatcgatg atatcagatc cactagtggc
     3061 ctatgcggcc gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca
     3121 ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3181 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3241 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3301 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3361 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3421 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3481 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3541 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3601 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3661 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3721 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3781 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3841 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3901 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3961 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4021 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4081 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4141 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4201 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4261 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     4321 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     4381 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     4441 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     4501 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     4561 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     4621 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     4681 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4741 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4801 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4861 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4921 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4981 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5041 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5101 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5161 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     5221 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     5281 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     5341 agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa
     5401 ttaatacata accttatgta tcatacacat acgatttagg tgacactata ga
//
