LOCUS       sequence-438163-        5104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for generating integration cassette to C-terminally tag gene
            of interest with pHluorin::HPHMX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5104)
  AUTHORS   Oppenheimer KG, Hager NA, McAtee CK, Filiztekin E, Shang C, Warnick 
            JA, Bruchez MP, Brodsky JL, Prosser DC, Kwiatkowski AV, O'Donnell AF
  TITLE     Optimization of the fluorogen-activating protein tag for 
            quantitative protein trafficking and colocalization studies in S. 
            cerevisiae.
  JOURNAL   Mol Biol Cell. 2024 Jul 1;35(7):mr5. doi: 10.1091/mbc.E24-04-0174. 
            Epub 2024 May 29.
  PUBMED    38809589
REFERENCE   2  (bases 1 to 5104)
  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: "Mol Biol 
            Cell."; date: "2024-07-1"; volume: "35(7):mr5. doi"; pages: " 
            10.1091/mbc.E24-04-0174. Epub 2024 May 29"
COMMENT     Sequence Label: pDP0437
FEATURES             Location/Qualifiers
     source          1..5104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             63..776
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             63..776
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Miesenbock et al., 1998)"
                     /label=ecliptic pHluorin
                     /note="responds to a drop in pH with a gradual loss 
                     (eclipsing) of excitation at 475 nm, accompanied by a 
                     reduction of excitation at 395 nm"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(90..118)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     713..733
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      797..984
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(852..876)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            1059..2634
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        1059..1402
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1403..2431
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1403..2431
                     /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      2437..2634
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(2741..2760)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2742..2760)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2847..2864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3018..3606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3098..3117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3777..4568,4569..4637))
                     /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     4400..4419
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4638..4742)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4810..4828
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4866..4888)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4988..5007
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5088..5104,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5088..5104,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 acagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
      121 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
      181 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
      241 tcactacttt cacttatggt gttcaatgct tttcaagata cccagatcat atgaaacggc
      301 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca
      361 aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta
      421 atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat
      481 tggaatacaa ctataacgat caccaggtgt acatcatggc agacaaacaa aagaatggaa
      541 tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg aggcgttcaa ctagcagacc
      601 attatcaaca aaatactcca attggcgatg ggcccgtcct tttaccagac aaccattacc
      661 tgtttacaac ttctactctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc
      721 ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaatagggcg
      781 cgccacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
      841 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
      901 attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagt atgaggtcgc
      961 tcttattgac cacacctcta ccggcagatc cgctagggat aacagggtaa tatagatctg
     1021 tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc cagaataccc
     1081 tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc ccgtacattt
     1141 agcccataca tccccatgta taatcatttg catccataca ttttgatggc cgcacggcgc
     1201 gaagcaaaaa ttacggctcc tcgctgcgga cctgcgagca gggaaacgct cccctcacag
     1261 acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt taggatttgc
     1321 cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac agttctcaca
     1381 tcacatccga acataaacaa ccatgggtaa aaagcctgaa ctcaccgcga cgtctgtcga
     1441 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     1501 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     1561 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     1621 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     1681 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     1741 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     1801 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     1861 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     1921 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     1981 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     2041 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     2101 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     2161 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     2221 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     2281 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     2341 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     2401 ccccagcact cgtccgaggg caaaggaata atcagtactg acaataaaaa gattcttgtt
     2461 ttcaagaact tgtcatttgt atagtttttt tatattgtag ttgttctatt ttaatcaaat
     2521 gttagcgtga tttatatttt ttttcgcctc gacatcatct gcccagatgc gaagttaagt
     2581 gcgcagaaag taatatcatg cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg
     2641 attcgatact aacgccgcca tccagtgtcg aaaacgagct cgaattcatc gatgatatca
     2701 gatccactag tggcctatgc ggccgcggat ctgccggtct ccctatagtg agtcgtatta
     2761 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2821 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2881 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2941 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3001 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3061 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3121 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3181 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3241 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3301 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3361 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3421 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3481 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3541 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3601 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3661 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3721 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3781 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3841 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3901 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3961 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4021 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4081 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4141 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4201 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4261 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4321 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4381 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4441 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4501 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4561 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4621 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4681 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4741 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4801 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     4861 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4921 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     4981 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     5041 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
     5101 tata
//
