LOCUS       sequence-326696-        6514 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 6514)
  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 6514)
  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..6514
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(42..629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(122..141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(800..1591,1592..1660))
                     /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     1423..1442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1661..1765)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1796..2175
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2177..2454
                     /label=chicken beta-actin promoter
     intron          2455..3467
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3391..3413
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3475..3494
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3528..3537
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      4089..4098
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4095..4811
                     /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(4128..4156)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4751..4771
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(5298..5317)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5363..5418
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5417..5436)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5809..5825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5809..5825)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5822..5844)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5833..5849
                     /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(5857..5863,5864..5881,5882..5887))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5902..5923
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5982..6177
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6028..6163
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6183..6317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6220..6239)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6274..6293
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6385..6402)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
       61 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      121 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      181 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      241 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      301 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      361 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      421 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      481 agacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
      541 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
      601 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
      661 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
      721 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
      781 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
      841 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
      901 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
      961 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     1021 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     1081 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     1141 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     1201 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     1261 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     1321 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     1381 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     1441 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     1501 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     1561 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     1621 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     1681 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     1741 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcgacat
     1801 tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat
     1861 atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     1921 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
     1981 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     2041 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     2101 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     2161 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     2221 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     2281 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
     2341 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
     2401 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     2461 gctgcgttgc cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc gccccggctc
     2521 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     2581 aattagcgct tggtttaatg acggctcgtt tcttttctgt ggctgcgtga aagccttaaa
     2641 gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt
     2701 gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc
     2761 ggcgcggggc tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc
     2821 cgcggtgcgg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     2881 gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc
     2941 gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc
     3001 tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc
     3061 gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag
     3121 gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc
     3181 ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg
     3241 ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg
     3301 cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg
     3361 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     3421 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg
     3481 tgctggttat tgtgctgtct catcattttg gcaaagaatt gctcgaggcc accatggacg
     3541 tggtgaatca gctggtggct gggggtcagt tccgggtggt caaggagccc cttggcttcg
     3601 tgaaggtgct gcagtgggtc tttgccatct tcgcctttgc tacgtgcggc agctacaccg
     3661 gagagcttcg gctgagcgtg gagtgtgcca acaagacgga gagtgccctc aacatcgaag
     3721 tcgaatttga gtacccattc aggctgcacc aagtgtactt tgatgcaccc tcctgcgtta
     3781 aagggggcac taccaagatc ttcctagttg gtgactactc ctcctcggct gaattctttg
     3841 tcaccgtggc tgtgtttgcc ttcctctact ccatgggggc cctggccacc tacatcttcc
     3901 tgcagaacaa gtaccgagag aacaacaaag ggccaatgat ggacttcctg gccacagcag
     3961 tgttcgcttt catgtggcta gttagctcat ccgcctgggc caaaggcctg tccgatgtga
     4021 agatggccac tgacccagag aacattatca aggagatgcc tatgtgccgc cagacagata
     4081 tggatctagc caccatggga agtaaaggag aagaactttt cactggagtt gtcccaattc
     4141 ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag
     4201 gtgatgcaac atacggaaaa cttaccctta aatttatttg cactactgga aaactacctg
     4261 ttccttggcc aacacttgtc actactttaa cttatggtgt tcaatgcttt tcaagatacc
     4321 cagatcatat gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg
     4381 aaagaactat atttttcaaa gatgacggga actacaagac acgtgctgaa gtcaagtttg
     4441 aaggtgatac ccttgttaat agaatcgagt taaaaggtat tgattttaaa gaagatggaa
     4501 acattcttgg acacaaattg gaatacaact ataacgatca ccaggtgtac atcatggcag
     4561 acaaacaaaa gaatggaatc aaagctaact tcaaaattag acacaacatt gaagatggag
     4621 gcgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggg cccgtccttt
     4681 taccagacaa ccattacctg tttacaactt ctactctttc gaaagatccc aacgaaaaga
     4741 gagaccacat ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg
     4801 aactatacaa aaccggtgga gctcgaatca catgcaagga actgagggac cctgtgactt
     4861 caggactcaa cacctcggtg gtgtttggct tcctgaacct ggtgctctgg gttggcaacc
     4921 tatggttcgt gttcaaggag acaggctggg ccgccccatt catgcgcgca cctccaggcg
     4981 ccccagaaaa gcaaccagct cctggcgatg cctacggcga tgcgggctat gggcagggcc
     5041 ccggaggcta tgggccccag gactcctacg ggcctcaggg tggttatcaa cccgattacg
     5101 ggcagccagc cagcggcggt ggcggtggct acgggcctca gggcgactat gggcagcaag
     5161 gctacggcca acagggtgcg cccacctcct tctccaatca gatgtaagcg gccgcaattc
     5221 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
     5281 cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc
     5341 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     5401 aattttttgt gtctatcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
     5461 aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa
     5521 ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
     5581 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
     5641 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
     5701 tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcaccgt
     5761 cgaccagctg gtcgacggtg caccgtcgac cagcttggcg taatcatggt catagctgtt
     5821 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5881 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5941 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     6001 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     6061 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     6121 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     6181 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     6241 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     6301 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6361 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     6421 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6481 taacgcagga aagaacatgt gagcaaaagg ccag
//
