LOCUS       sequence-304807-        2751 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  gateway donor vector encoding ratiometric sensor of lysosomal 
            lumenal pH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2751)
  AUTHORS   Ponsford AH, Ryan TA, Raimondi A, Cocucci E, Wycislo SA, Frohlich F,
            Swan LE, Stagi M
  TITLE     Live imaging of intra-lysosome pH in cell lines and primary neuronal
            culture using a novel genetically encoded biosensor.
  JOURNAL   Autophagy. 2020 Jun 9:1-19. doi: 10.1080/15548627.2020.1771858.
  PUBMED    32515674
REFERENCE   2  (bases 1 to 2751)
  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. 
            2020 Jun 9:1-19. doi: 10.1080/15548627.2020.1771858."
COMMENT     Sequence Label: pDONOR223  R2pH-LAMP1-3xFLAG
FEATURES             Location/Qualifiers
     source          1..2751
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             82..789
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(227..245)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     495..514
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             808..1521
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(838..866)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1461..1481
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2677..2742
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
ORIGIN
        1 atggcggccc ccggcgcccg gcggccgctg ctcctgctgc tgctggcagg ccttgcacat
       61 ggcgcctcag cagctagcgg tatggtgagc aagggcgagg aggataacat ggccatcatc
      121 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
      181 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
      241 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
      301 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
      361 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
      421 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
      481 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
      541 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
      601 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
      661 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
      721 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
      781 ctgtacaagg ctagcggggg gcccgggatg agtaaaggag aagaactttt cactggagtt
      841 gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga
      901 gagggtgaag gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga
      961 aaactacctg ttccatggcc aacacttgtc actactttaa cttatggtgt tcaatgcttt
     1021 tcaagatacc cagatcatat gaaacggcat gactttttca agagtgccat gcccgaaggt
     1081 tatgtacagg aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa
     1141 gtcaagtttg aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa
     1201 gaagatggaa acattcttgg acacaaattg gaatacaact ataacgatca ccaggtgtac
     1261 atcatggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattag acacaacatt
     1321 gaagatggag gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggg
     1381 cccgtccttt taccagacaa ccattacctg tttacaactt ctactctttc gaaagatccc
     1441 aacgaaaaga gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat
     1501 ggcatggatg aactatacaa acccgggggg ctctttgagg tgaaaaacaa tggcacaacg
     1561 tgtataatgg ccagcttctc tgcctccttt ctgaccacct acgagactgc gaatggttct
     1621 cagatcgtga acatttccct gccagcctct gcagaagtac tgaaaaatgg cagttcttgt
     1681 ggtaaagaaa atgtttctga ccccagcctc acaattactt ttggaagagg atatttactg
     1741 acactcaact tcacaaaaaa tacaacacgt tacagtgtcc agcatatgta ttttacatat
     1801 aacttgtcag atacagaaca ttttcccaat gccatcagca aagagatcta caccatggat
     1861 tccacaactg acatcaaggc agacatcaac aaagcatacc ggtgtgtcag tgatatccgg
     1921 gtctacatga agaatgtgac cgttgtgctc cgggatgcca ctatccaggc ctacctgtcg
     1981 agtggcaact tcagcaagga agagacacac tgcacacagg atggaccttc cccaaccact
     2041 gggccaccca gcccctcacc accacttgtg cccacaaacc ccactgtatc caagtacaat
     2101 gttactggta acaacggaac ctgcctgctg gcctctatgg cactgcaact gaatatcacc
     2161 tacctgaaaa aggacaacaa gacggtgacc agagcgttca acatcagccc aaatgacaca
     2221 tctagtggga gttgcggtat caacttggtg accctgaaag tggagaacaa gaacagagcc
     2281 ctggaattgc agtttgggat gaatgccagc tctagcctgt ttttcctgca aggagtgcgc
     2341 ttgaatatga ctcttcctga tgccctagtg cccacattca gcatctccaa ccattcactg
     2401 aaagctcttc aggccactgt gggaaactca tacaagtgca acactgagga acacatcttt
     2461 gtcagcaaga tgctctccct caatgtcttc agtgtgcagg tccaggcttt caaggtggac
     2521 agtgacaggt ttgggtctgt ggaagagtgt gttcaggatg gtaacaacat gttgatcccc
     2581 attgctgtgg gcggtgccct ggcagggctg gtcctcatcg tcctcattgc ctacctcatt
     2641 ggcaggaaga ggagtcacgc cggctatcag accatcgact acaaagacca tgacggtgat
     2701 tataaagatc atgacatcga ctacaaggat gacgatgaca agtaggaatt c
//
