LOCUS       sequence-323570-        5336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of lyso-pHluorin in mammalian cells for optical readout 
            of lysosomal acidification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5336)
  AUTHORS   Rost BR, Schneider F, Grauel MK, Wozny C, G Bentz C, Blessing A, 
            Rosenmund T, Jentsch TJ, Schmitz D, Hegemann P, Rosenmund C
  TITLE     Optogenetic acidification of synaptic vesicles and lysosomes.
  JOURNAL   Nat Neurosci. 2015 Nov 9. doi: 10.1038/nn.4161.
  PUBMED    26551543
REFERENCE   2  (bases 1 to 5336)
  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: "Nat 
            Neurosci. 2015 Nov 9. doi: 10.1038/nn.4161."
COMMENT     Sequence Label: pCMV-lyso-pHluorin
FEATURES             Location/Qualifiers
     source          1..5336
                     /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      738..747
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             744..1460
                     /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="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(777..805)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1400..1420
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2064..2090
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2124..2245
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2161..2180)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2215..2234
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2252..2707)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2389..2410)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2601..2620
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2734..2838
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2840..3197
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2840..3031
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2863..2883)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3048..3183
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3232..4026
                     /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             3232..4026
                     /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(3286..3305)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3896..3915
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4214..4233)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4258..4305
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4634..5222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5123..5142
                     /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 cttggcagta 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 gctagcacca
      601 tggcggtgga aggaggaatg aagtgtgtca agtttttgct ctacgttctc ctgctggcct
      661 tctgcgcctg tgcagtggga ttgatcgcca ttggtgtagc ggttcaggtt gtcttgaagc
      721 aggccgaagc ggatctagcc accatgggaa gtaaaggaga agaacttttc actggagttg
      781 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
      841 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
      901 aactacctgt tccttggcca acacttgtca ctactttaac ttatggtgtt caatgctttt
      961 caagataccc agatcatatg aaacggcatg actttttcaa gagtgccatg cccgaaggtt
     1021 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
     1081 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
     1141 aagatggaaa cattcttgga cacaaattgg aatacaacta taacgatcac caggtgtaca
     1201 tcatggcaga caaacaaaag aatggaatca aagctaactt caaaattaga cacaacattg
     1261 aagatggagg cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatgggc
     1321 ccgtcctttt accagacaac cattacctgt ttacaacttc tactctttcg aaagatccca
     1381 acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg
     1441 gcatggatga actatacaaa accggtggag ctccaattac ccatgagact actgctggct
     1501 cgctgttgcc tgtggtcatc attgcagtgg gtgccttcct cttcctggtg gcctttgtgg
     1561 gctgctgtgg ggcctgcaag gagaactact gtctcatgat tacatttgcc atcttcctgt
     1621 ctcttatcat gcttgtggag gtggctgtgg ccattgctgg ctatgtgttt agagaccagg
     1681 tgaagtcaga gtttaataaa agcttccagc agcagatgca gaattacctt aaagacaaca
     1741 aaacagccac tattttggac aaattgcaga aagaaaataa ctgctgtgga gcttctaact
     1801 acacagactg ggaaaacatc cccggcatgg ccaaggacag agtccccgat tcttgctgca
     1861 tcaacataac tgtgggctgt gggaatgatt tcaaggaatc cactatccat acccagggct
     1921 gcgtggagac tatagcaata tggctaagga agaacatact gctggtggct gcagcggccc
     1981 tgggcattgc ttttgtggag gtcttgggaa ttatcttctc ctgctgtctg gtgaagagta
     2041 ttcgaagtgg ctatgaagta atgtacccat acgatgttcc agattacgct tagttaatta
     2101 agcaagggcg aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2161 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2221 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     2281 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2341 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2401 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2461 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2521 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2581 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2641 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2701 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2761 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2821 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     2881 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2941 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3001 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3061 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3121 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3181 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3241 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3301 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3361 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3421 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3481 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3541 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3601 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3661 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3721 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     3781 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3841 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3901 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3961 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4021 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4081 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4141 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4201 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4261 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4321 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     4381 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     4441 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4501 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4741 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5281 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
