LOCUS       sequence-324281-        6702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lysosomal pH biosensor consisting of sfGFP-rat Lamp1-mCherry..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6702)
  AUTHORS   Webb BA, Aloisio FM, Charafeddine RA, Cook J, Wittmann T, Barber DL
  TITLE     pHLARE: A New Biosensor Reveals Decreased Lysosome pH in Cancer 
            Cells.
  JOURNAL   Mol Biol Cell. 2020 Nov 25:mbcE20060383. doi: 
            10.1091/mbc.E20-06-0383.
  PUBMED    33237838
REFERENCE   2  (bases 1 to 6702)
  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. 2020 Nov 25:mbcE20060383. doi: 10.1091/mbc.E20-06-0383."
COMMENT     Sequence Label: psfGFP-LAMP1-mCherry (pHLARE)
FEATURES             Location/Qualifiers
     source          1..6702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      620..629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             626..715
                     /codon_start=1
                     /product="signal sequence from bovine prolactin"
                     /label=prolactin signal sequence
                     /translation="MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS"
     CDS             716..1426
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(755..776)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1363..1384
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      2657..2666
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2663..3370
                     /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(2808..2826)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3076..3095
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3490..3611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3527..3546)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3581..3600
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3618..4073)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3755..3776)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3967..3986
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4100..4204
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4206..4563
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4206..4397
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4229..4249)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4414..4549
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4598..5392
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4598..5392
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4652..4671)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5262..5281
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5580..5599)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5624..5671
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6000..6588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6489..6508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttagcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagcg ccaccatgga cagcaaaggt tcgtcgcaga aagggtcccg
      661 cctgctcctg ctgctggtgg tgtcaaatct actcttgtgc cagggtgtgg tctccagcaa
      721 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
      781 cggccacaag ttcagcgtgc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac
      841 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
      901 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc gccacgactt
      961 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcagct tcaaggacga
     1021 cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     1081 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     1141 caacttcaac agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     1201 caacttcaag atccgccaca acgtggagga cggcagcgtg cagctcgccg accactacca
     1261 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     1321 ccagtccgtg ctgagcaaag accccaacga gaagcgcgac cacatggtcc tgctggagtt
     1381 cgtgaccgcc gccgggatca ctcacggcat ggacgagctg tacaagtccg gattgagcgc
     1441 caccgcccca gcactgttcg aggtgaaaga caacaacggc acagcgtgta taatggccag
     1501 cttctctgcc tcctttctga ccacctatga ggctggacat gtttctaagg tctcgaatat
     1561 gaccctgcca gcctctgcag aagtcctgaa gaatagcagc tcttgtggtg aaaagaatgc
     1621 ttctgagccc accctcgcaa tcacctttgg agaaggatat ttactgaaac tcaccttcac
     1681 aaaaaacaca acacgttaca gtgtccagca catgtatttc acatataacc tgtcagacac
     1741 acaattcttt cccaatgcca gctccaaagg gcccgacact gtggattcca caactgacat
     1801 caaggcagac atcaacaaaa cataccgatg tgtcagcgac atcagggtct acatgaagaa
     1861 tgtgaccatt gtgctctggg acgctactat ccaggcctac ctgccgagta gcaacttcag
     1921 caaggaagag acacgctgcc cacaggatca accttcccca actactgggc cacccagccc
     1981 ctcaccacca cttgtgccca caaaccccag tgtgtccaag tacaatgtga ctggtgacaa
     2041 tggaacctgc ctgctggcct ctatggcact gcaactcaac atcacctaca tgaagaagga
     2101 caacacgact gtgaccagag cattcaacat caacccaagt gacaaatata gtgggacttg
     2161 cggtgcccag ttggtgaccc tgaaggtggg gaacaagagc agagtcctgg agctgcagtt
     2221 tgggatgaat gccacttcta gcctgttttt cctgcaagga gttcagttga acatgactct
     2281 tcctgatgcc atagagccca cgttcagcac ctccaactat tccctgaaag ctcttcaggc
     2341 cagtgtcggc aactcataca agtgcaacag tgaggagcac atctttgtca gcaaggcgct
     2401 cgccctcaat gtcttcagcg tgcaagtcca ggctttcagg gtagaaagtg acaggtttgg
     2461 gtctgtggaa gagtgtgtac aggacggtaa caacatgctg atccccattg ctgtgggcgg
     2521 ggccctggca gggctggtcc tcatcgtcct catcgcctat ctcatcggca ggaagaggag
     2581 tcacgcgggc tatcagacca tctcggaatt cggctccacc ggctccaccg gctccaccgg
     2641 cgcggatcca ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat
     2701 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     2761 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     2821 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     2881 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     2941 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     3001 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     3061 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     3121 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     3181 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     3241 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     3301 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     3361 gctgtacaaa gtaagcgact ctagatcata atcagccata ccacatttgt agaggtttta
     3421 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3481 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3541 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3601 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3661 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3721 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3781 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     3841 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3901 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3961 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4021 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     4081 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4141 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4201 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     4261 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     4321 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4381 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4441 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4501 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4561 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4621 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4681 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4741 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     4801 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4861 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4921 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4981 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     5041 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     5101 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     5161 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     5221 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     5281 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     5341 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     5401 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5461 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5521 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     5581 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     5641 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5701 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     5761 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     5821 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     5881 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5941 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6001 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6061 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6121 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6181 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6241 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6301 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6361 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6421 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6481 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6541 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6601 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6661 ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
