LOCUS       sequence-378602-        5626 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (pH-sensitive) super ecliptic pHluorin-tagged rat Vamp3 
            for imaging exocytosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5626)
  AUTHORS   Lam M, Takeo K, Almeida RG, Cooper MH, Wu K, Iyer M, Kantarci H, 
            Zuchero JB
  TITLE     CNS myelination requires VAMP2/3-mediated membrane expansion in 
            oligodendrocytes.
  JOURNAL   Nat Commun. 2022 Sep 23;13(1):5583. doi: 10.1038/s41467-022-33200-4.
  PUBMED    36151203
REFERENCE   2  (bases 1 to 5626)
  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 
            Commun."; date: "2022-09-23"; volume: "13(1):5583. doi"; pages: " 
            10.1038/s41467-022-33200-4"
COMMENT     Sequence Label: pCMV-Vamp3-pHluorin
FEATURES             Location/Qualifiers
     source          1..5626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   13..142
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        218..521
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        522..725
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     676..696
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     722..746
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          874..1252
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1651..2367
                     /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(1681..1709)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2304..2324
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2385..2861
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2584..2603)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2901..3041
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2901..3030
                     /label=AAV2 ITR
     rep_origin      3116..3571
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3203..3222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3413..3434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3588..3607)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3707..3729
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3767..3785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3853..3957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3958..4026,4027..4818)
                     /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     complement(4176..4195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4989..5577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5478..5497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
       61 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
      121 aactccatca ctaggggttc ctgcggccgc acgcgtctag ttattaatag taatcaatta
      181 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      241 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      301 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      361 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      421 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      481 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      541 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      601 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      661 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      721 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
      781 atagaagaca ccgggaccga tccagcctcc gcggattcga atcccggccg ggaacggtgc
      841 attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg
      901 cccacaaaaa atgctttctt cttttaatat acttttttgt ttatcttatt tctaatactt
      961 tccctaatct ctttctttca gggcaataat gatacaatgt atcatgcctc tttgcaccat
     1021 tctaaagaat aacagtgata atttctgggt taaggcaata gcaatatttc tgcatataaa
     1081 tatttctgca tataaattgt aactgatgta agaggtttca tattgctaat agcagctaca
     1141 atccagctac cattctgctt ttattttatg gttgggataa ggctggatta ttctgagtcc
     1201 aagctaggcc cttttgctaa tcatgttcat acctcttatc ttcctcccac agctcctggg
     1261 caacgtgctg gtctgtgtgc tggcccatca ctttggcaaa gaattgggat tcgaacatcg
     1321 attgaattcg ccaccatgtc tacaggggtg ccttcagggt ccagtgctgc cactggcagt
     1381 aatcgaagac tccagcagac acagaatcaa gtagatgagg tggtggacat catgagagtc
     1441 aatgtggata aggtgttaga aagagaccag aagctctcag agctagacga ccgcgcagat
     1501 gcactgcagg caggcgcctc gcagtttgaa acaagtgctg ccaagttgaa gagaaagtat
     1561 tggtggaaga actgcaagat gtgggcgata gggatcagtg tcctggtgat tattgtcatt
     1621 atcatcattg tgtggtgtgt ctcttccgga atgagtaaag gagaagaact tttcactgga
     1681 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
     1741 ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat ttgcactact
     1801 ggaaaactac ctgttccttg gccaacactt gtcactactt taacttatgg tgttcaatgc
     1861 ttttcaagat acccagatca tatgaaacgg catgactttt tcaagagtgc catgcccgaa
     1921 ggttatgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa gacacgtgct
     1981 gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg tattgatttt
     2041 aaagaagatg gaaacattct tggacacaaa ttggaataca actataacga tcaccaggtg
     2101 tacatcatgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat tagacacaac
     2161 attgaagatg gaggcgttca actagcagac cattatcaac aaaatactcc aattggcgat
     2221 gggcccgtcc ttttaccaga caaccattac ctgtttacaa cttctactct ttcgaaagat
     2281 cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca
     2341 catggcatgg atgaactata caaataatga ctcgagagat ctacgggtgg catccctgtg
     2401 acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc accagccttg
     2461 tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat aatattatgg
     2521 ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt agggcctgcg
     2581 gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact gcaatctccg
     2641 cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga ttccaggcat
     2701 gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc accatattgg
     2761 ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct cccaaattgc
     2821 tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg taggtaacca
     2881 cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     2941 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3001 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     3061 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     3121 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3181 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3241 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3301 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     3361 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3421 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     3481 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3541 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     3601 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3661 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3721 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     3781 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     3841 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3901 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3961 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4021 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4081 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4141 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4201 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4261 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4321 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4381 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4441 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4501 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4561 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4621 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4681 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4741 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4801 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4861 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4921 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4981 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5041 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5101 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5161 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5221 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5281 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5341 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5401 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5461 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5521 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5581 gccagcaacg cggccttttt acggttcctg gccttttgct ggcctt
//
