LOCUS       sequence-321888-        4939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  plasmid for homologous recombination-mediated insertion of triple HA
            tag into synaptophysin gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4939)
  AUTHORS   Chantranupong L, Saulnier JL, Wang W, Jones DR, Pacold ME, Sabatini 
            BL
  TITLE     Rapid purification and metabolomic profiling of synaptic vesicles 
            from mammalian brain.
  JOURNAL   Elife. 2020 Oct 12;9. pii: 59699. doi: 10.7554/eLife.59699.
  PUBMED    33043885
REFERENCE   2  (bases 1 to 4939)
  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: "Elife."; 
            date: "2020-10-12"; volume: "9. pii: 59699. doi"; pages: " 
            10.7554/eLife.59699"
COMMENT     Sequence Label: pAAV-HDR-sphys-3XHA
FEATURES             Location/Qualifiers
     source          1..4939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        149..397
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     149..169
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     320..339
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        419..494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1459..1485
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1498..1524
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1537..1563
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     repeat_region   2202..2342
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2202..2331
                     /label=AAV2 ITR
     rep_origin      2417..2872
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2504..2523)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2714..2735
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2889..2908)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3008..3030
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3068..3086)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3154..3258
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3259..3327,3328..4119)
                     /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(3477..3496)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4290..4878
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4779..4798
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gaaagcttga gggcctattt cccatgattc cttcatattt
      181 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
      241 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
      301 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
      361 cttggcttta tatatcttgt ggaaaggacg aaacaccgtt ctccaatcag atgtaatcgt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt gaattctttt ggttttgttt gagacaggat ctacttatgt
      541 gaccctggct gtcctggaac tcactactca gaccagactg gcctcaaact cacagacctc
      601 tgcttgcctc tgcctcctga gtactaagat gaagactgca ccaccacacc cagcccaaaa
      661 atgagttgtt tgaggctgac tttcatgttg cacaggctag cctcaaacta tgaatttaaa
      721 ggtagacttg aatttctggg tagtggaggc agagacaggc gacttctatg agttccaggc
      781 cagcctggtc tacagagtga attccaggac agccagggct gcagagagac cctgtctcaa
      841 aaaaaaaaag ctagccttga agtgatcgcc cctgcctcca gcttcctaag attacaagat
      901 gtgggccttc agacttgtcc atgtaaacac tgatagaagt tgaacatcat gggaatctaa
      961 cacacacaca cacactccca agtttttctg tacactgata gtcatagagg cccacgaatt
     1021 tatgccctaa aaatgcccat tcctgttcac tcagcctcaa agaccctggg gctgccgagg
     1081 caatgggtaa gagacaacag ctttggtcat gtctccctgc aggtgtttgg cttcctgaac
     1141 ctggtgctct gggttggcaa cctatggttc gtgttcaagg agacaggctg ggccgcccca
     1201 ttcatgcgcg cacctccagg cgccccagaa aagcaaccag ctcctggcga tgcctacggc
     1261 gatgcgggct atgggcaggg ccccggaggc tatgggcccc aggactccta cgggcctcag
     1321 ggtggttatc aacccgatta cgggcagcca gccagcggcg gtggcggtgg ctacgggcct
     1381 cagggcgact atgggcagca aggctacggc caacagggtg cgcccacctc cttctccaac
     1441 caaatgggag ggagcggcta tccctatgac gtgcctgatt acgccggcac aggatcctac
     1501 ccctatgatg tgcctgacta cgctggcagc gccggatacc cttatgatgt gcctgattat
     1561 gcttaatctg gtgagtgaca actgggcgga tgcggtaggc agggagcata caaggagtga
     1621 agtttgaagg aaccaataga taggcagaac caaaaaaaaa aaaaggtgaa cttggtaaaa
     1681 ctagccaatg agaggaaccg tgagaaggaa ggggacggag cagtgctcag agtaaccaat
     1741 gaaaggagtg taggggcact tgcgcagtgg agaatcacca aagtggtgta ggtttccagg
     1801 aagggaaggg gaggagggtc tttgaaatca ttggtaaacc aataggcggt aacgccagta
     1861 ggtggaagaa ggtaaacacg ttgggttttg aagggcgcta gcgctaaagc aggatgtagg
     1921 tcagctgcta cctctcctta accctttaat gaaagagaga gtttggaatt tcaaatgagg
     1981 aaaaggggag ggctggaggc cttagaaaca cgagtatgcc tttttgttgg gcctttaaaa
     2041 aatgaatgcc gccggacggt ggaggcgcac gcctttaatc ccagcacttg ggaggcagaa
     2101 gcaggcggag tttttgagtt cgaggccagc ctggtctaca aagtgagttc caggacagcc
     2161 agggctatac agagaaaccc tgtcgcggtc gacgcggccg caggaacccc tagtgatgga
     2221 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     2281 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     2341 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     2401 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2461 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     2521 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     2581 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     2641 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2701 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     2761 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     2821 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     2881 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     2941 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3001 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     3061 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     3121 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3181 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3241 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3301 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3361 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3421 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3481 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3541 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3601 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3661 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3721 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3781 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3841 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3901 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3961 gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4021 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4081 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4141 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4201 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4261 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4321 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4381 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4441 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     4501 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     4561 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     4621 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     4681 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     4741 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     4801 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     4861 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     4921 tggccttttg ctcacatgt
//
