LOCUS       sequence-378601-        5683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses (pH-sensitive) super ecliptic pHluorin-tagged mouse/rat 
            Vamp2 for imaging exocytosis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5683)
  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 5683)
  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-Vamp2-pHluorin
FEATURES             Location/Qualifiers
     source          1..5683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..134
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        210..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        514..717
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     668..688
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     714..738
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          866..1244
                     /label=chimeric intron
                     /note="fusion between human cytomegalovirus intron A and 
                     human beta-globin intron 2"
     CDS             1703..2416
                     /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(1730..1758)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2353..2373
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2434..2910
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2633..2652)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2950..3090
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2950..3079
                     /label=AAV2 ITR
     rep_origin      3165..3620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3252..3271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3462..3483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3637..3656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3756..3778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3816..3834)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3902..4006
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4007..4075,4076..4867)
                     /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(4225..4244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5038..5626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5527..5546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
       61 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      121 cactaggggt tcctgcggcc gcacgcgtct agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      541 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      601 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      661 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      721 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      781 caccgggacc gatccagcct ccgcggattc gaatcccggc cgggaacggt gcattggaac
      841 gcggattccc cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacaaa
      901 aaatgctttc ttcttttaat atactttttt gtttatctta tttctaatac tttccctaat
      961 ctctttcttt cagggcaata atgatacaat gtatcatgcc tctttgcacc attctaaaga
     1021 ataacagtga taatttctgg gttaaggcaa tagcaatatt tctgcatata aatatttctg
     1081 catataaatt gtaactgatg taagaggttt catattgcta atagcagcta caatccagct
     1141 accattctgc ttttatttta tggttgggat aaggctggat tattctgagt ccaagctagg
     1201 cccttttgct aatcatgttc atacctctta tcttcctccc acagctcctg ggcaacgtgc
     1261 tggtctgtgt gctggcccat cactttggca aagaattggg attcgaacat cgattgaatt
     1321 cgccaccatg tcggctaccg ctgccaccgt cccgcctgcc gccccggccg gcgagggtgg
     1381 cccccctgca cctcctccaa accttactag taacaggaga ctgcagcaga cccaggccca
     1441 ggtggatgag gtggtggaca tcatgagggt gaatgtggac aaggtcctgg agcgggacca
     1501 gaagttgtcg gagctggatg accgtgcaga tgccctccag gcaggggcct cccagtttga
     1561 aacaagtgca gccaagctca agcgcaaata ctggtggaaa aacctcaaga tgatgatcat
     1621 cttgggagtg atctgcgcca tcatcctcat catcatcatc gtttacttca gcactagcgg
     1681 cggaagcggc gggaccggtg gaagtaaagg agaagaactt ttcactggag ttgtcccaat
     1741 tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga
     1801 aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc
     1861 tgttccttgg ccaacacttg tcactacttt aacttatggt gttcaatgct tttcaagata
     1921 cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca
     1981 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt
     2041 tgaaggtgat acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg
     2101 aaacattctt ggacacaaat tggaatacaa ctataacgat caccaggtgt acatcatggc
     2161 agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg
     2221 aggcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg ggcccgtcct
     2281 tttaccagac aaccattacc tgtttacaac ttctactctt tcgaaagatc ccaacgaaaa
     2341 gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga
     2401 tgaactatac aaataatgac tcgagagatc tacgggtggc atccctgtga cccctcccca
     2461 gtgcctctcc tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa
     2521 ttaagttgca tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg
     2581 gtggtatgga gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg
     2641 gaaccaagct ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt
     2701 caagcgattc tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg
     2761 ctcagctaat ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc
     2821 tccaactcct aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag
     2881 gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg
     2941 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3001 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3061 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     3121 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     3181 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3241 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3301 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3361 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3421 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3481 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     3541 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3601 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     3661 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     3721 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     3781 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     3841 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3901 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3961 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4021 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4081 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4141 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4201 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4261 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4321 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4381 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4441 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     4501 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     4561 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     4621 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     4681 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     4741 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     4801 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     4861 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4921 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4981 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5041 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5101 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5161 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5221 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5281 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5341 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5401 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5461 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5521 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5581 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5641 ggccttttta cggttcctgg ccttttgctg gccttttgct cac
//
