LOCUS       sequence-490855-        6517 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  To endogenously tag S. cerevisiae proteins at their C-terminus with 
            Pho8∆60; K.l.LEU2 selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6517)
  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 6517)
  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-K.I.LEU2
FEATURES             Location/Qualifiers
     source          1..6517
                     /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"
     primer_bind     complement(4152..4171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4153..4171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4258..4275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4429..5017)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4509..4528)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5188..5979,5980..6048))
                     /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     5811..5830
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6049..6153)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6221..6239
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6277..6299)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6399..6418
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6499..6517
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6499..6516
                     /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 atccgctgtg aagatcccag caaaggctta caaagtgtta
     1861 tctcttttga gacttgttga gttgaacact ggtgttttca tcaaacttac caaggacgtg
     1921 tacccattgt tgaaacttgt atcaccatat attgttatcg gacaaccttc acttgcatct
     1981 atccgttctt taatccaaaa gagatctaga ataatgtggc aaaggccaga agataaagaa
     2041 ccaaaagaga taatcttgaa tgacaacaat atcgttgaag agaaattagg tgatgaaggt
     2101 gtcatttgta tcgaggatat catccatgag atttcgacgt tgggcgaaaa tttctcgaaa
     2161 tgtactttct tcctattacc attcaaattg aacagagaag tcagtggatt cggtgccatc
     2221 tcccgtttga ataaactgaa aatgcgcgaa caaaacaaga agactcgtca aatttcaaac
     2281 gctgccacgg ctccagttat ccaagtagat atcgactcaa tgatttccaa gttgaattga
     2341 ttaactataa aaggaaaata tctgtacaat agacatcggg ctcccattgg ccctacccac
     2401 atatgtagaa atacattact ctattcacta ctgcatttag ttatgtttaa catttgatat
     2461 agcagacgct accgccaggc acaatatatt ccccttccct cttgccattc gctgtacttg
     2521 tggtagattc caattcagcg cagtcacgtg ctagtaatca ccgcattttt ttcttttcct
     2581 ttcaggctaa aaccggttcc gggcctgatc cctgcactca ttttctaacg gaaaaccttc
     2641 agaagcataa ctacccattc cagtttagag tcatgacagg ttcaacatca gatgcttcat
     2701 atacttttat atattgaatt atataaatat atctatgtac tctaagtaag tacatctgct
     2761 ttaacgcatt cctacatttg cttcgattta tttttattgt tgatacctat ttgaagaagt
     2821 aaaaagtatc ccacactaca cagattatac catgtctaag aatatcgttg tcctaccggg
     2881 tgatcacgtc ggtaaagaag ttactgacga agctattaag gtcttgaatg ccattgctga
     2941 agtccgtcca gaaattaagt tcaatttcca acatcacttg atcgggggtg ctgccatcga
     3001 tgccactggc actcctttac cagatgaagc tctagaagcc tctaagaaag ccgatgctgt
     3061 cttactaggt gctgttggtg gtccaaaatg gggtacgggc gcagttagac cagaacaagg
     3121 tctattgaag atcagaaagg aattgggtct atacgccaac ttgagaccat gtaactttgc
     3181 ttctgattct ttactagatc tttctccttt gaagcctgaa tatgcaaagg gtaccgattt
     3241 cgtcgtcgtt agagaattgg ttggtggtat ctactttggt gaaagaaaag aagatgaagg
     3301 tgacggagtt gcttgggact ctgagaaata cagtgttcct gaagttcaaa gaattacaag
     3361 aatggctgct ttcttggcat tgcaacaaaa cccaccatta ccaatctggt ctcttgacaa
     3421 ggctaacgtg cttgcctctt ccagattgtg gagaaagact gttgaagaaa ccatcaagac
     3481 tgagttccca caattaactg ttcagcacca attgatcgac tctgctgcta tgattttggt
     3541 taaatcacca actaagctaa acggtgttgt tattaccaac aacatgtttg gtgatattat
     3601 ctccgatgaa gcctctgtta ttccaggttc tttgggttta ttaccttctg catctctagc
     3661 ttccctacct gacactaaca aggcattcgg tttgtacgaa ccatgtcatg gttctgcccc
     3721 agatttacca gcaaacaagg ttaacccaat tgctaccatc ttatctgcag ctatgatgtt
     3781 gaagttatcc ttggatttgg ttgaagaagg tagggctctt gaagaagctg ttagaaatgt
     3841 cttggatgca ggtgtcagaa ccggtgacct tggtggttct aactctacca ctgaggttgg
     3901 cgatgctatc gccaaggctg tcaaggaaat cttggcttaa agagtctttt gtaacgaccc
     3961 cgtctccacc aacttggtat gcttgaaatc tcaaggccat tacacattca gttatgtgaa
     4021 cgaaaggtct ttatttaacg tagcataaac taaataatac aggttccggt tagcctgcgt
     4081 ttaaacgagc tcgaattcat cgatgatatc agatccacta gtggcctatg cggccgcgga
     4141 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     4201 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4261 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4321 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4381 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4441 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4501 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4561 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4621 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4681 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4741 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4801 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4861 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4921 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4981 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5041 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5101 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5161 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5221 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5281 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5341 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5401 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5461 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5521 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5581 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5641 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5701 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5761 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5821 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5881 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5941 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6001 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6061 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6121 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6181 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     6241 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     6301 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     6361 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     6421 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt
     6481 atgtatcata cacatacgat ttaggtgaca ctataga
//
