LOCUS       sequence-318961-        7428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses ratiometric sensor of lysosomal lumenal pH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7428)
  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 7428)
  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: pCAG RpH-LAMP1-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          541..1557
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1481..1503
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1565..1584
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1613..1622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1696..2403
                     /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(1841..1859)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2109..2128
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2425..3138
                     /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(2455..2483)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3078..3098
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             4294..4359
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4446..4465)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4511..4566
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4565..4584)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4927..4943)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4927..4943)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4940..4962)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4951..4967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4975..4981,4982..4999,5000..5005))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5020..5041
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5100..5295
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5146..5281
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5301..5435
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5338..5357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5392..5411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5503..5520)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5674..6262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5754..5773)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6433..7224,7225..7293))
                     /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     7056..7075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7294..7398)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatcg
      421 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      481 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      541 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      601 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      661 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      721 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      781 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      841 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
      901 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
      961 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1021 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1081 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1141 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1201 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1261 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1321 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1381 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1441 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1501 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1561 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt caccatggcg
     1621 gcccccggcg cccggcggcc gctgctcctg ctgctgctgg caggccttgc acatggcgcc
     1681 tcagcagcta gcggtatggt gagcaagggc gaggaggata acatggccat catcaaggag
     1741 ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag
     1801 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     1861 ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag
     1921 gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc
     1981 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac
     2041 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     2101 tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg
     2161 taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc
     2221 ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc
     2281 ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc
     2341 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     2401 aagggagcta gcggggggcc cgggatgagt aaaggagaag aacttttcac tggagttgtc
     2461 ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag
     2521 ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa
     2581 ctacctgttc cttggccaac acttgtcact actttaactt atggcgttca atgcttttca
     2641 agatacccag atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat
     2701 gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc
     2761 aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa
     2821 gatggaaaca ttcttggaca caaattggaa tacaactata acgatcacca ggtgtacatc
     2881 atggcagaca aacaaaagaa tggaatcaaa gctaacttca aaattagaca caacattgaa
     2941 gatggaggcg ttcaactagc agaccattat caacaaaata ctccaattgg cgatgggccc
     3001 gtccttttac cagacaacca ttacctgttt acaacttcta ctctttcgaa agatcccaac
     3061 gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc
     3121 atggatgaac tatacaaacc cggggggctc tttgaggtga aaaacaatgg cacaacgtgt
     3181 ataatggcca gcttctctgc ctcctttctg accacctacg agactgcgaa tggttctcag
     3241 atcgtgaaca tttccctgcc agcctctgca gaagtactga aaaatggcag ttcttgtggt
     3301 aaagaaaatg tttctgaccc cagcctcaca attacttttg gaagaggata tttactgaca
     3361 ctcaacttca caaaaaatac aacacgttac agtgtccagc atatgtattt tacatataac
     3421 ttgtcagata cagaacattt tcccaatgcc atcagcaaag agatctacac catggattcc
     3481 acaactgaca tcaaggcaga catcaacaaa gcataccggt gtgtcagtga tatccgggtc
     3541 tacatgaaga atgtgaccgt tgtgctccgg gatgccacta tccaggccta cctgtcgagt
     3601 ggcaacttca gcaaggaaga gacacactgc acacaggatg gaccttcccc aaccactggg
     3661 ccacccagcc cctcaccacc acttgtgccc acaaacccca ctgtatccaa gtacaatgtt
     3721 actggtaaca acggaacctg cctgctggcc tctatggcac tgcaactgaa tatcacctac
     3781 ctgaaaaagg acaacaagac ggtgaccaga gcgttcaaca tcagcccaaa tgacacatct
     3841 agtgggagtt gcggtatcaa cttggtgacc ctgaaagtgg agaacaagaa cagagccctg
     3901 gaattgcagt ttgggatgaa tgccagctct agcctgtttt tcctgcaagg agtgcgcttg
     3961 aatatgactc ttcctgatgc cctagtgccc acattcagca tctccaacca ttcactgaaa
     4021 gctcttcagg ccactgtggg aaactcatac aagtgcaaca ctgaggaaca catctttgtc
     4081 agcaagatgc tctccctcaa tgtcttcagt gtgcaggtcc aggctttcaa ggtggacagt
     4141 gacaggtttg ggtctgtgga agagtgtgtt caggatggta acaacatgtt gatccccatt
     4201 gctgtgggcg gtgccctggc agggctggtc ctcatcgtcc tcattgccta cctcattggc
     4261 aggaagagga gtcacgccgg ctatcagacc atcgactaca aagaccatga cggtgattat
     4321 aaagatcatg acatcgacta caaggatgac gatgacaagt aggaattcac tcctcaggtg
     4381 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     4441 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     4501 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     4561 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     4621 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     4681 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     4741 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     4801 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     4861 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     4921 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4981 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5041 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     5101 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     5161 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5221 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     5281 ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca
     5341 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     5401 ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg
     5461 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5521 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5581 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5641 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5701 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5761 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5821 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5881 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5941 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6001 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6061 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6121 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6181 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6241 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6301 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6361 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6421 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6481 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6541 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6601 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6661 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6721 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6781 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6841 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6901 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6961 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7021 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7081 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7141 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7201 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7261 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7321 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7381 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtc
//
