LOCUS       sequence-354972-        5530 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a pH insensitive blue fluorencent protein mTagBFP fused to 
            the N-terminal of a membrane targeted pH sensitive green fluorescent
            protein pHluorin-CAAX under CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5530)
  AUTHORS   Wu L, Dong A, Dong L, Wang SQ, Li Y
  TITLE     PARIS, an optogenetic method for functionally mapping gap junctions.
  JOURNAL   Elife. 2019 Jan 14;8. pii: 43366. doi: 10.7554/eLife.43366.
  PUBMED    30638447
REFERENCE   2  (bases 1 to 5530)
  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: "Elife."; 
            date: "2019-01-14"; volume: "8. pii: 43366. doi"; pages: " 
            10.7554/eLife.43366"
COMMENT     Sequence Label: pN3-mTagBFP-pHluorin-CAAX
FEATURES             Location/Qualifiers
     source          1..5530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        121..424
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        425..628
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     579..599
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    669..725
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      729..738
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1494..2204
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(1521..1549)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2144..2164
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2378..2499
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2415..2434)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2469..2488
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2506..2961)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2643..2664)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2855..2874
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2988..3092
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3094..3451
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3094..3285
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3117..3137)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3302..3437
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3486..4280
                     /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             3486..4280
                     /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(3540..3559)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4150..4169
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4468..4487)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4512..4559
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4888..5476
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5377..5396
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
       61 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      121 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      181 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      241 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      301 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      361 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      421 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      481 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      541 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      601 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      661 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      721 gatccatcgc caccatggtg agcaagggcg aggagagcga ggaactcatc aaagaaaaca
      781 tgcatatgaa actgtacatg gagggaaccg tggataatca ccacttcaag tgtactagtg
      841 agggcgaggg caaaccttac gagggaaccc agaccatgag aatcaaagtc gtggagggcg
      901 gacctctgcc ttttgctttc gacattctgg ccacatcctt tctctacggc tccaaaacct
      961 tcatcaacca cacacagggc atccccgatt ttttcaaaca gagctttcct gagggattca
     1021 cctgggagcg agtgacaaca tacgaggacg gaggagtgct caccgctaca caggacacat
     1081 cactccagga tggatgtctc atctacaacg tgaaaatccg aggcgtgaac ttcacttcca
     1141 atggccccgt catgcagaaa aaaaccctgg gctgggaggc ttttactgaa acactctacc
     1201 ctgccgatgg cgggctcgag gggcgaaatg acatggctct caaactggtc ggcggatcac
     1261 atctcattgc caatatcaaa accacctacc ggtctaaaaa acctgccaaa aacctcaaaa
     1321 tgcccggcgt ctactacgtc gattaccggc tggaacggat caaagaggcc aacaacgaaa
     1381 cctacgtgga acagcacgaa gtcgccgtgg ctcggtactg tgacctcccc tctaaactcg
     1441 ggcacaaact gaacggcatg gacgagctgt acaagggcgg cagcggcggc gtgagtaaag
     1501 gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg
     1561 ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc
     1621 ttaaatttat ttgcactact ggaaaactac ctgttccttg gccaacactt gtcactactt
     1681 taacttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacgg catgactttt
     1741 tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc aaagatgacg
     1801 ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg
     1861 agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa ttggaataca
     1921 actataacga tcaccaggtg tacatcatgg cagacaaaca aaagaatgga atcaaagcta
     1981 acttcaaaat tagacacaac attgaagatg gaggcgttca actagcagac cattatcaac
     2041 aaaatactcc aattggcgat gggcccgtcc ttttaccaga caaccattac ctgtttacaa
     2101 cttctactct ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg
     2161 taacagctgc tgggattaca catggcatgg atgaactata caaagatggt aaaaagaaga
     2221 aaaagaagtc aaagacaaag tgtgtaatta tgtaaagcgg ccgcgactct agatcataat
     2281 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2341 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2401 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2461 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     2521 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2581 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     2641 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     2701 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     2761 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2821 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2881 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2941 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3001 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     3061 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     3121 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3181 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3241 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3301 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3361 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3421 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     3481 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3541 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3601 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3661 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3721 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3781 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3841 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3901 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3961 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     4021 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4081 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4141 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4201 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4261 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4321 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4381 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4441 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     4501 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     4561 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     4621 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     4681 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     4741 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4801 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4861 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4921 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4981 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5041 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5101 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5161 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5221 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5281 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5341 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5401 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5461 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5521 gccttttgct
//
