LOCUS       sequence-418643-        6964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of lyso-pHoenix in mammalian cells for optogenetic 
            acidification of lysosomes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6964)
  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 6964)
  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-pHoenix
FEATURES             Location/Qualifiers
     source          1..6964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             116..142
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    176..297
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(213..232)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     267..286
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(304..759)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(441..462)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     653..672
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        786..890
                     /gene="bla"
                     /label=AmpR promoter
     promoter        892..1249
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        892..1083
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(915..935)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1100..1235
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1284..2078
                     /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             1284..2078
                     /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(1338..1357)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1948..1967
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2266..2285)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2310..2357
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2686..3274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3175..3194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3560..3763
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3714..3734
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3933..3942
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3939..4655
                     /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(3972..4000)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4595..4615
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             4671..5444
                     /codon_start=1
                     /product="light-driven proton pump archaerhodopsin-3 from 
                     Halorubrum sodomense (Chow et al., 2010)"
                     /label=Arch
                     /translation="MDPIALQAGYDLLGDGRPETLWLGIGTLLMLIGTFYFLVRGWGVT
                     DKDAREYYAVTILVPGIASAAYLSMFFGIGLTEVTVGGEMLDIYYARYADWLFTTPLLL
                     LDLALLAKVDRVTIGTLVGVDALMIVTGLIGALSHTAIARYSWWLFSTICMIVVLYFLA
                     TSLRSAAKERGPEVASTFNTLTALVLVLWTAYPILWIIGTEGAGVVGLGIETLLFMVLD
                     VTAKVGFGFILLRSRAILGDTEAPEPSAGADVSAAD"
     CDS             5451..6146
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
ORIGIN
        1 gaagaacata ctgctggtgg ctgcagcggc cctgggcatt gcttttgtgg aggtcttggg
       61 aattatcttc tcctgctgtc tggtgaagag tattcgaagt ggctatgaag taatgtaccc
      121 atacgatgtt ccagattacg cttagttaat taagcaaggg cgaattgttg ttgttaactt
      181 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
      241 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
      301 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
      361 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
      421 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
      481 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
      541 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
      601 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
      661 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
      721 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
      781 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      841 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
      901 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
      961 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     1021 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     1081 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     1141 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     1201 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     1261 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     1321 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     1381 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     1441 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     1501 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     1561 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     1621 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     1681 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     1741 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     1801 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     1861 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     1921 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     1981 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     2041 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     2101 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     2161 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     2221 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     2281 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     2341 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     2401 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     2461 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     2521 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2581 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2641 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2701 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2761 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2821 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2881 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2941 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3001 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3061 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3121 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3181 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3241 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3301 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3361 tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat
     3421 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     3481 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     3541 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     3601 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     3661 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     3721 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     3781 gatccgctag caccatggcg gtggaaggag gaatgaagtg tgtcaagttt ttgctctacg
     3841 ttctcctgct ggccttctgc gcctgtgcag tgggattgat cgccattggt gtagcggttc
     3901 aggttgtctt gaagcaggcc gaagcggatc tagccaccat gggaagtaaa ggagaagaac
     3961 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
     4021 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
     4081 tttgcactac tggaaaacta cctgttcctt ggccaacact tgtcactact ttaacttatg
     4141 gtgttcaatg cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg
     4201 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
     4261 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     4321 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataacg
     4381 atcaccaggt gtacatcatg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
     4441 ttagacacaa cattgaagat ggaggcgttc aactagcaga ccattatcaa caaaatactc
     4501 caattggcga tgggcccgtc cttttaccag acaaccatta cctgtttaca acttctactc
     4561 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     4621 ctgggattac acatggcatg gatgaactat acaaaaccgg taccggatcc atggacccca
     4681 tcgctctgca ggctggttac gacctgctgg gtgacggcag acctgaaact ctgtggctgg
     4741 gcatcggcac tctgctgatg ctgattggaa ccttctactt tctggtccgc ggatggggag
     4801 tcaccgataa ggatgcccgg gaatattacg ctgtgactat cctggtgccc ggaatcgcat
     4861 ccgccgcata tctgtctatg ttctttggta tcgggcttac tgaggtgacc gtcgggggcg
     4921 aaatgttgga tatctattat gccaggtacg ccgactggct gtttaccacc ccacttctgc
     4981 tgctggatct ggcccttctc gctaaggtgg atcgggtgac catcggcacc ctggtgggtg
     5041 tggacgccct gatgatcgtc actggcctca tcggagcctt gagccacacg gccatagcca
     5101 gatacagttg gtggttgttc tctacaattt gcatgatagt ggtgctctat tttctggcta
     5161 catccctgcg atctgctgca aaggagcggg gccccgaggt ggcatctacc tttaacaccc
     5221 tgacagctct ggtcttggtg ctgtggaccg cttaccctat cctgtggatc ataggcactg
     5281 agggcgctgg cgtggtgggc ctgggcatcg aaactctgct gtttatggtg ttggacgtga
     5341 ctgccaaggt cggctttggc tttatcctgt tgagatcccg ggctattctg ggcgacaccg
     5401 aggcaccaga acccagtgcc ggtgccgatg tcagtgccgc cgaccgtacg atggtgagcg
     5461 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg aacaaccacc
     5521 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     5581 tcaaggcggt cgagggcggc cctctcccct tcgccttcga catcctggct accagcttca
     5641 tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc tttaagcagt
     5701 ccttccccga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     5761 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     5821 gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     5881 ccaccgagac cctgtacccc gctgacggcg gcctggaagg cagagccgac atggccctga
     5941 agctcgtggg cgggggccac ctgatctgca acttgaagac cacatacaga tccaagaaac
     6001 ccgctaagaa cctcaagatg cccggcgtct actatgtgga cagaagactg gaaagaatca
     6061 aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc agatactgcg
     6121 acctccctag caaactgggg cacagaaagc ttgcagccct gcaggagaag aagtcatgca
     6181 gccagcgcat ggccgaattc cggcaatact gttggaaccc ggacactggg cagatgctgg
     6241 gccgcacccc agcccggtgg gtgtggatca gcctgtacta tgcagctttc tacgtggtca
     6301 tgactgggct ctttgccttg tgcatctatg tgctgatgca gaccattgat ccctacaccc
     6361 ccgactacca ggaccagtta aagtcaccgg gggtaacctt gagaccggat gtgtatgggg
     6421 aaagagggct gcagatttcc tacaacatct ctgaaaacag ctctggttta aacaccaccg
     6481 gtggagctcc aattacccat gagactactg ctggctcgct gttgcctgtg gtcatcattg
     6541 cagtgggtgc cttcctcttc ctggtggcct ttgtgggctg ctgtggggcc tgcaaggaga
     6601 actactgtct catgattaca tttgccatct tcctgtctct tatcatgctt gtggaggtgg
     6661 ctgtggccat tgctggctat gtgtttagag accaggtgaa gtcagagttt aataaaagct
     6721 tccagcagca gatgcagaat taccttaaag acaacaaaac agccactatt ttggacaaat
     6781 tgcagaaaga aaataactgc tgtggagctt ctaactacac agactgggaa aacatccccg
     6841 gcatggccaa ggacagagtc cccgattctt gctgcatcaa cataactgtg ggctgtggga
     6901 atgatttcaa ggaatccact atccataccc agggctgcgt ggagactata gcaatatggc
     6961 taag
//
