LOCUS       sequence-330214-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Synapto-pHluorin for optical monitoring of presynaptic
            release in neurons. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  AUTHORS   Kwon SK, Sando R 3rd, Lewis TL, Hirabayashi Y, Maximov A, Polleux F
  TITLE     LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance 
            and Neurotransmitter Release Properties at Excitatory Synapses along
            Cortical Axons.
  JOURNAL   PLoS Biol. 2016 Jul 18;14(7):e1002516. doi: 
            10.1371/journal.pbio.1002516. eCollection 2016 Jul.
  PUBMED    27429220
REFERENCE   2  (bases 1 to 6473)
  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: "PLoS 
            Biol."; date: "2016-07-18"; volume: "14(7):e1002516. doi"; pages: " 
            10.1371/journal.pbio.1002516. eCollection 2016 Jul"
COMMENT     Sequence Label: pCAG-Synapto-pHluorin
FEATURES             Location/Qualifiers
     source          1..6473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(80..668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(160..179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(839..1630,1631..1699))
                     /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"
     primer_bind     1462..1481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1700..1804)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1835..2214
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2448..3464
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3388..3410
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3472..3491
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3525..3534
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4086..4095
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4092..4808
                     /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(4125..4153)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4748..4768
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(5295..5314)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5360..5415
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5414..5433)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5806..5822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5806..5822)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5819..5841)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5830..5846
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5854..5860,5861..5878,5879..5884))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5899..5920
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5979..6174
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6025..6160
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6180..6314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6217..6236)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6271..6290
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6382..6399)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
       61 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
      121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
      541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
      781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
      841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
      901 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
      961 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     1021 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     1081 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     1141 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     1201 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     1261 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     1321 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     1381 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     1441 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     1501 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     1561 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     1621 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     1681 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     1741 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     1801 cgcgcacatt tccccgaaaa gtgccacctg ggtcgacatt gattattgac tagttattaa
     1861 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     1921 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     1981 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     2041 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     2101 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     2161 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag
     2221 gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg
     2281 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg ggggcgaggc
     2341 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
     2401 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     2461 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     2521 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     2581 agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc
     2641 tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg
     2701 tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg
     2761 cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc
     2821 ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt
     2881 gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag
     2941 ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg
     3001 ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg
     3061 ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg
     3121 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt
     3181 tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc
     3241 gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt
     3301 cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct
     3361 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     3421 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     3481 tggttattgt gctgtctcat cattttggca aagaattgct cgaggccacc atggacgtgg
     3541 tgaatcagct ggtggctggg ggtcagttcc gggtggtcaa ggagcccctt ggcttcgtga
     3601 aggtgctgca gtgggtcttt gccatcttcg cctttgctac gtgcggcagc tacaccggag
     3661 agcttcggct gagcgtggag tgtgccaaca agacggagag tgccctcaac atcgaagtcg
     3721 aatttgagta cccattcagg ctgcaccaag tgtactttga tgcaccctcc tgcgttaaag
     3781 ggggcactac caagatcttc ctagttggtg actactcctc ctcggctgaa ttctttgtca
     3841 ccgtggctgt gtttgccttc ctctactcca tgggggccct ggccacctac atcttcctgc
     3901 agaacaagta ccgagagaac aacaaagggc caatgatgga cttcctggcc acagcagtgt
     3961 tcgctttcat gtggctagtt agctcatccg cctgggccaa aggcctgtcc gatgtgaaga
     4021 tggccactga cccagagaac attatcaagg agatgcctat gtgccgccag acagatatgg
     4081 atctagccac catgggaagt aaaggagaag aacttttcac tggagttgtc ccaattcttg
     4141 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
     4201 atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc
     4261 cttggccaac acttgtcact actttaactt atggtgttca atgcttttca agatacccag
     4321 atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa
     4381 gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag
     4441 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca
     4501 ttcttggaca caaattggaa tacaactata acgatcacca ggtgtacatc atggcagaca
     4561 aacaaaagaa tggaatcaaa gctaacttca aaattagaca caacattgaa gatggaggcg
     4621 ttcaactagc agaccattat caacaaaata ctccaattgg cgatgggccc gtccttttac
     4681 cagacaacca ttacctgttt acaacttcta ctctttcgaa agatcccaac gaaaagagag
     4741 accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac
     4801 tatacaaaac cggtggagct cgaatcacat gcaaggaact gagggaccct gtgacttcag
     4861 gactcaacac ctcggtggtg tttggcttcc tgaacctggt gctctgggtt ggcaacctat
     4921 ggttcgtgtt caaggagaca ggctgggccg ccccattcat gcgcgcacct ccaggcgccc
     4981 cagaaaagca accagctcct ggcgatgcct acggcgatgc gggctatggg cagggccccg
     5041 gaggctatgg gccccaggac tcctacgggc ctcagggtgg ttatcaaccc gattacgggc
     5101 agccagccag cggcggtggc ggtggctacg ggcctcaggg cgactatggg cagcaaggct
     5161 acggccaaca gggtgcgccc acctccttct ccaatcagat gtaagcggcc gcaattcact
     5221 cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac
     5281 aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt
     5341 gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat
     5401 tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat
     5461 gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt
     5521 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat
     5581 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct
     5641 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga
     5701 ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct gcaccgtcga
     5761 ccagctggtc gacggtgcac cgtcgaccag cttggcgtaa tcatggtcat agctgtttcc
     5821 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5881 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5941 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     6001 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     6061 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     6121 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     6181 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     6241 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     6301 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6361 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6421 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat cag
//
