LOCUS       sequence-489660-        5576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To endogenously tag S. cerevisiae proteins at their C-terminus with 
            Pho8∆60; HIS3 selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5576)
  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 5576)
  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"; 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-His3MX6
FEATURES             Location/Qualifiers
     source          1..5576
                     /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        1864..2207
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2208..2906
                     /codon_start=1
                     /product="S. kluyveri imidazoleglycerol phosphate 
                     dehydratase"
                     /label=k-HIS3
                     /translation="MAEPAQKKQKQTVQERKAFISRITNETKIQIAISLNGGYIQIKDS
                     ILPAKKDDDVASQATQSQVIDIHTGVGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHT
                     TEDCGIALGQAFKEAMGAVRGVKRFGTGFAPLDEALSRAVVDLSSRPFAVIDLGLKREM
                     IGDLSTEMIPHFLESFAEAARITLHVDCLRGFNDHHRSESAFKALAVAIREAISSNGTN
                     DVPSTKGVLM"
     terminator      2910..3107
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(3211..3230)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3212..3230)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3317..3334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3488..4076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3568..3587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4247..5038,5039..5107))
                     /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     4870..4889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5108..5212)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5280..5298
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5336..5358)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5458..5477
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5558..5576
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5558..5575
                     /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 tccttttaaa
     2161 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg gcagaaccag
     2221 cccaaaaaaa gcaaaaacaa actgttcagg agcgcaaggc gtttatctcc cgtatcacta
     2281 atgaaactaa aattcaaatc gctatttcgc tgaatggtgg ttatattcaa ataaaagatt
     2341 cgattcttcc tgcaaagaag gatgacgatg tagcttccca agctactcag tcacaggtca
     2401 tcgatattca cacaggtgtt ggctttttgg atcatatgat ccatgcgttg gcaaaacact
     2461 ctggttggtc tcttattgtt gaatgtattg gtgacctgca cattgacgat caccatacta
     2521 ccgaagattg cggtatcgca ttagggcaag cgttcaaaga agcaatgggt gctgtccgtg
     2581 gtgtaaaaag attcggtact gggttcgcac cattggatga ggcgctatca cgtgccgtag
     2641 tcgatttatc tagtagacca tttgctgtaa tcgaccttgg attgaagaga gagatgattg
     2701 gtgatttatc cactgaaatg attccacact ttttggaaag tttcgcggag gcggccagaa
     2761 ttactttgca tgttgattgt ctgagaggtt tcaacgatca ccacagaagt gagagtgcgt
     2821 tcaaggcttt ggctgttgcc ataagagaag ctatttctag caatggcacc aatgacgttc
     2881 cctcaaccaa aggtgttttg atgtgaagta ctgacaataa aaagattctt gttttcaaga
     2941 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     3001 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     3061 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat
     3121 actaacgccg ccatccagtt taaacgagct cgaattcatc gatgatatca gatccactag
     3181 tggcctatgc ggccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     3241 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3301 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3361 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3421 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3481 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3541 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3601 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3661 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3721 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3781 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3841 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3901 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3961 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4021 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4081 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4141 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4201 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4261 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4321 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4381 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4441 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4501 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4561 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4621 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4681 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4741 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4801 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4861 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4921 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4981 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5041 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5101 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5161 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5221 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5281 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     5341 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     5401 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     5461 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct
     5521 acaattaata cataacctta tgtatcatac acatacgatt taggtgacac tataga
//
