LOCUS       sequence-369487-        6235 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Membrane targeted cytoplasmic fluorescent pH sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6235)
  AUTHORS   Armbruster M, Naskar S, Garcia JP, Sommer M, Kim E, Adam Y, Haydon 
            PG, Boyden ES, Cohen AE, Dulla CG
  TITLE     Neuronal activity drives pathway-specific depolarization of 
            peripheral astrocyte processes.
  JOURNAL   Nat Neurosci. 2022 May;25(5):607-616. doi: 
            10.1038/s41593-022-01049-x. Epub 2022 Apr 28.
  PUBMED    35484406
REFERENCE   2  (bases 1 to 6235)
  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"; doi: 
            "10.1038/s41593-022-01049-x"; journalName: "Nat Neurosci"; date: 
            "2022-05"; volume: "25"; issue: "5"; pages: "607-616"
COMMENT     Sequence Label: GFAP-Lyn-mCherry-pHluorin
FEATURES             Location/Qualifiers
     source          1..6235
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             1002..1709
                     /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(1147..1165)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1415..1434
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1722..2438
                     /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(1752..1780)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2375..2395
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2471..2512
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    2530..3118
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2583..2603)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3001..3012)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3143..3160
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3183..3200)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3189..3413
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3498..3638
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3498..3627
                     /label=AAV2 ITR
     rep_origin      3713..4168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3800..3819)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4010..4031
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4185..4204)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4304..4326
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4364..4382)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4450..4554
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4555..4623,4624..5415)
                     /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"
     rep_origin      5586..6174
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6075..6094
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcaattc agtcgataac tataacggtc ctaaggtagc gatttaaata
      181 cgcgctctct taaggtagcc ccgggacgcg tcaattgaga acatatcctg gtgtggagta
      241 ggggacgctg ctctgacaga ggctcggggg cctgagctgg ctctgtgagc tggggaggag
      301 gcagacagcc aggccttgtc tgcaagcaga cctggcagca ttgggctggc cgccccccag
      361 ggcctcctct tcatgcccag tgaatgactc accttggcac agacacaatg ttcggggtgg
      421 gcacagtgcc tgcttcccgc cgcaccccag cccccctcaa atgccttccg agaagcccat
      481 tgagcagggg gcttgcattg caccccagcc tgacagcctg gcatcttggg ataaaagcag
      541 cacagccccc taggggctgc ccttgctgtg tggcgccacc ggcggtggag aacaaggctc
      601 tattcagcct gtgcccagga aaggggatca ggggatgccc aggcatggac agtgggtggc
      661 agggggggag aggagggctg tctgcttccc agaagtccaa ggacacaaat gggtgagggg
      721 actggagggg agctctcccc atagctgggc tgcggcccaa ccccaccccc tcaggctatg
      781 ccagggggtg ttgccagggg cacccgggca tcgccagtct agcccactcc ttcataaagc
      841 cctcgcatcc caggagcgag cagagccaga gcaggttgga gaggagacgc atcacctccg
      901 ctgctcgccg ctagcgttta aacttaagct tggtaccgag ctcggatctc gagagaaata
      961 tgggatgtat taaatcaaaa aggaaagacg ggagaattcc catggtgagc aagggcgagg
     1021 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     1081 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     1141 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     1201 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     1261 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     1321 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     1381 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg
     1441 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     1501 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     1561 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     1621 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     1681 ccaccggcgg catggacgag ctgtacaagg gatccgccac catgagtaaa ggagaagaac
     1741 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
     1801 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
     1861 tttgcactac tggaaaacta cctgttcctt ggccaacact tgtcactact ttaacttatg
     1921 gtgttcaatg cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg
     1981 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
     2041 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     2101 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataacg
     2161 atcaccaggt gtacatcatg gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa
     2221 ttagacacaa cattgaagat ggaggcgttc aactagcaga ccattatcaa caaaatactc
     2281 caattggcga tgggcccgtc cttttaccag acaaccatta cctgtttaca acttctactc
     2341 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     2401 ctgggattac acatggcatg gatgaactat acaaataaag cggccgctcg agtctagagg
     2461 gcccttcgaa ggtaagccta tccctaaccc tctcctcggt ctcgattcta cgcgtaccgg
     2521 ttaatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     2581 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     2641 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     2701 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     2761 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     2821 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     2881 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     2941 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     3001 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     3061 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     3121 cgaaacccgc tgatcagccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc
     3181 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3241 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3301 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3361 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3421 ggcggaaaga accagatcct ctcttaaggt agcatcgaga tttaaattag ggataacagg
     3481 gtaatggcgc gggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     3541 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     3601 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     3661 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     3721 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3781 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3841 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3901 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3961 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4021 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     4081 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4141 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4201 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4261 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4321 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4381 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4441 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4501 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     4561 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     4621 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     4681 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     4741 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     4801 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     4861 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     4921 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     4981 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5041 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5101 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5161 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5221 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     5281 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5341 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5401 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5461 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5521 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5581 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5641 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5701 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     5761 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5821 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5881 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5941 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6001 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6061 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6121 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6181 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
