LOCUS       sequence-378606-        6589 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dominant-negative Vamp3 (rat Vamp3 AA 1-81) plus 
            membrane-targeted EGFP in oligodendrocytes; AAV vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6589)
  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 6589)
  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: AAV_pMBP-dnVamp3-P2AT2A-EGFP-caax
FEATURES             Location/Qualifiers
     source          1..6589
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   20..149
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             2423..2479
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2489..2542
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2549..2551,2552..2554,2555..3265)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2549..2551,2552..2554,2555..3265)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2594..2615)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2855..2874)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3202..3223
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3355..3831
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3554..3573)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3871..4011
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3871..4000
                     /label=AAV2 ITR
     rep_origin      4086..4541
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4173..4192)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4383..4404
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4558..4577)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4677..4699
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4737..4755)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4823..4927
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4928..4996,4997..5788)
                     /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(5146..5165)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5959..6547
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6448..6467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac tgaggccgcc
       61 cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg
      121 agtggccaac tccatcacta ggggttcctg cggccgcacg cgtgagctcc ttcctgctta
      181 ggccgtgtct gctctgtcag tctttcctct ggctctagtc tgtccatctg cagggataac
      241 ctcccaaggg ccctcttgtt ggctgttcac cttccgtctg tcttcatagc ctcctgatga
      301 gagagcgcgc acacacaggc tacctgactg atgtgttgct gcactgtggg gtccagtgac
      361 acagctgtgg aggtcaggaa cccagaatta agctgtctgg gagccacaag tttcaagagt
      421 ccatgctatg gctcctacat gcaggtggtg ccagcagcat tccttggtgt cctgtggctg
      481 tggttgcatc tctgactctc ctccacctat cacattcctt ctccagtacc aggcctctgt
      541 gtctcagttt tcctctctca caagatccct agcttcagga ttgcacccac ctccactagc
      601 catgagcccg tcctcactgg atcacgtctg caaagactct atttccagtt aaagtcacag
      661 acacgggcat agagaggggg ataggatatg aaaatatcag ttgggggagg ggcattcaat
      721 tcttagctca cattttcttc atccaatttc ccccactaga atgtaagctt tgagagaaaa
      781 gggaccagat cttattcctc accgtggctt taacacttag agaaaatgca tcccctctaa
      841 tcaataagtc atcgacagtg ggtagatgga ggaacggcag tgcgtagtag gatgcgtgca
      901 agcatagtct cgtgcatggg tgcatagatc gctgggcagg tggacaaggt gggggtggat
      961 aaagaagtgg gtagatgatt gatgttaggt aaatatcact gggtggacag atgggtggta
     1021 ggtggatgga tggttagaat agtcagaaga gggatggatt gataaggtga acagatgata
     1081 aatgggtgat agactggaag ggttgtcaaa agaggataag ggaagtgtga gctagccgta
     1141 tttctaaggt cagtaataga gttgggagaa gaggttaagt tacatccatt taaacctcac
     1201 acgaagctga gagggaatgg acttgctgcc gttggtgagg aaagcgttgc atttcccgtg
     1261 tgcttggttg tgaagtgctc aggtcccaca tgaagcagtc aggttactgc ggcttacaga
     1321 ggagccagat ccaaatgccc cgagtaagca cgtccccgag ccagaggcct ccagcggaat
     1381 ccgggagagg gattgctcag tgccctgctt ccctggactg taagctgcag aaagatgtgg
     1441 gaagtcctgt tctccactga gaacactaaa agcacctttt gtcaaacgac cgcttcacat
     1501 ctggggcttg tgcactggtg gccttttaaa ccagagacaa cccacaagat acctaacctg
     1561 cggggctctc tggtacagtg agcaactcag gaaatgcttt ggcttgattg ctgtgggctc
     1621 tcaggccatc gccctctgga gtggttcttt taatgagaac ctgaagattg gcccctgagc
     1681 catgtatacc aagcaagctc aatccaggtt agctccctct ggttggggca agctaacgtg
     1741 ctccttgggc cccgcgcgta actgtgcgtt ttataggaga cagctagttc aagaccccag
     1801 gaagaaagcg gctttgtccc cctctaggcc tcgtacaggc ccacattcat atctcattgt
     1861 tgttgcaggg gaggcagatg cgatccagaa caatgggacc tcggctgagg acacggcggt
     1921 gacagactcc aagcacacag cagacccaaa gaataactgg caaggcgccc acccagctga
     1981 cccagggaac cgcccccact tgatccgcct cttttcccga gatgccccgg gaagggagga
     2041 caacaccttc aaagacaggc cctcagagtc cgacgagctt cagaccatcc aagaagaccc
     2101 cacagcagct tccggaagct cgaataattc taggatccga acatcgattg aattcgccac
     2161 catgtctaca ggggtgcctt cagggtccag tgctgccact ggcagtaatc gaagactcca
     2221 gcagacacag aatcaagtag atgaggtggt ggacatcatg agagtcaatg tggataaggt
     2281 gttagaaaga gaccagaagc tctcagagct agacgaccgc gcagatgcac tgcaggcagg
     2341 cgcctcgcag tttgaaacaa gtgctgccaa gttgaagaga aagtattggt ggaagaactg
     2401 caagaccggt agcggcggaa gcgctactaa cttcagcctg ctgaagcagg ctggagacgt
     2461 ggaggagaac cctggacctg gaagcggaga gggcagagga agtctgctaa catgcggtga
     2521 cgtcgaggag aatcctggac ctgctagcat ggtgagcaag ggcgaggagc tgttcaccgg
     2581 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
     2641 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
     2701 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg
     2761 cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
     2821 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
     2881 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
     2941 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     3001 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     3061 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     3121 cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga
     3181 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     3241 tctcggcatg gacgagctgt acaagtccgg actcagatct aagctgaacc ctcctgatga
     3301 gagtggcccc ggctgcatga gctgcaagtg tgtgctctcc tgataaagat ctacgggtgg
     3361 catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac tccagtgccc
     3421 accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt gtccttctat
     3481 aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa gacaacctgt
     3541 agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc ttggctcact
     3601 gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga gttgttggga
     3661 ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag acggggtttc
     3721 accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc accttggcct
     3781 cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct tctgattttg
     3841 taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag ttggccactc
     3901 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     3961 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4021 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4081 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4141 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     4201 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     4261 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     4321 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     4381 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     4441 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     4501 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     4561 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     4621 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     4681 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     4741 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     4801 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     4861 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     4921 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     4981 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5041 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5101 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     5161 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     5221 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     5281 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     5341 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     5401 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     5461 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     5521 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5581 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5641 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5701 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5761 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5821 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5881 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5941 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6001 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6061 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgc
//
