LOCUS       sequence-489658-        5918 bp    DNA     circular SYN 17-OCT-2025
DEFINITION  To endogenously tag S. cerevisiae proteins at their C-terminus with 
            Pho8∆60; HphMX6 selection marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 17, 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-HphMX6
FEATURES             Location/Qualifiers
     source          1..5918
                     /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..3236
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2208..3236
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      3244..3491
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3260..3278)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(3553..3572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3554..3572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3659..3676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3830..4418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3910..3929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4589..5380,5381..5449))
                     /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     5212..5231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5450..5554)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5622..5640
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5678..5700)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5800..5819
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5900..5918
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5900..5917
                     /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 gcggacctgc
     2041 gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag
     2101 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa
     2161 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaaaaagc
     2221 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     2281 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     2341 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     2401 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     2461 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     2521 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     2581 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     2641 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     2701 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     2761 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     2821 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     2881 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     2941 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     3001 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     3061 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     3121 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     3181 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataatctc
     3241 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     3301 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     3361 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     3421 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     3481 ctttaacgag ctcgaattca tcgatgatat cagatccact agtggcctat gcggccgcgg
     3541 atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt
     3601 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3661 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3721 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3781 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3841 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3901 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3961 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4021 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4081 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4141 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4201 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4261 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4321 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4381 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4441 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4501 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4561 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4621 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4681 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4741 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4801 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4861 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4921 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4981 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5041 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5101 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5161 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5221 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5281 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5341 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5401 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5461 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5521 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5581 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5641 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5701 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5761 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     5821 tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     5881 tatgtatcat acacatacga tttaggtgac actataga
//
