LOCUS       sequence-430692-        6404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To tag endogenous mouse VAMP2 with HA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6404)
  AUTHORS   Bindu DS, Tan CX, Savage JT, Eroglu C
  TITLE     GEARBOCS: An Adeno Associated Virus Tool for In Vivo Gene Editing in
            Astrocytes.
  JOURNAL   bioRxiv. 2023 Jan 19:2023.01.17.524433. doi: 
            10.1101/2023.01.17.524433. Preprint.
  PUBMED    36711516
REFERENCE   2  (bases 1 to 6404)
  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: "bioRxiv. 
            2023 Jan 19:2023.01.17.524433. doi: 10.1101/2023.01.17.524433. 
            Preprint."
COMMENT     Sequence Label: GEARBOCS-Vamp2-Tag-HA
FEATURES             Location/Qualifiers
     source          1..6404
                     /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        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        415..490
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             528..554
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     intron          1398..1530
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1513..1532
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1575..1593
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1614..1634
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3293..3414
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3304..3323)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3358..3377
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3576..3705
                     /label=AAV2 ITR
     primer_bind     complement(3724..3741)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3724..3740)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3733..3755)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3882..4337
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3969..3988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4179..4200
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4354..4373)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4473..4495
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4533..4551)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4619..4723
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4724..4792,4793..5584)
                     /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"
     rep_origin      5755..6343
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6244..6263
                     /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 gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgggccgg ggcggcaggc gggagtttta
      421 gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc
      481 gagtcggtgc ttttttggat ccggccgggg cggcaggcgg gacggattac ccttacgacg
      541 ttccagacta cgctacggcc ggggcggcag gcgggacggg tcgactctag actgcagagg
      601 gagatctaac atatcctggt gtggagtagg ggacgctgct ctgacagagg ctcgggggcc
      661 tgagctggct ctgtgagctg gggaggaggc agacagccag gccttgtctg caagcagacc
      721 tggcagcatt gggctggccg ccccccaggg cctcctcttc atgcccagtg aatgactcac
      781 cttggcacag acacaatgtt cggggtgggc acagtgcctg cttcccgccg caccccagcc
      841 cccctcaaat gccttccgag aagcccattg agcagggggc ttgcattgca ccccagcctg
      901 acagcctggc atcttgggat aaaagcagca cagcccccta ggggctgccc ttgctgtgtg
      961 gcgccaccgg cggtggagaa caaggctcta ttcagcctgt gcccaggaaa ggggatcagg
     1021 ggatgcccag gcatggacag tgggtggcag ggggggagag gagggctgtc tgcttcccag
     1081 aagtccaagg acacaaatgg gtgaggggag agctctcccc atagctgggc tgcggcccaa
     1141 ccccaccccc tcaggctatg ccagggggtg ttgccagggg cacccgggca tcgccagtct
     1201 agcccactcc ttcataaagc cctcgcatcc caggagcgag cagagccaga gcaggttgga
     1261 gaggagacgc atcacctccg ctgctcgcaa gctttattgc ggtagtttat cacagttaaa
     1321 ttgctaacgc agtcagtgct tctgacacaa cagtctcgaa cttaagctgc agaagttggt
     1381 cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga
     1441 aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta
     1501 ctgacatcca ctttgccttt ctctccacag gtgtccactc ccagttcaat tacagctctt
     1561 aaggctagag tacttaatac gactcactat aggctagcgg taccgccacc atgcccaaga
     1621 agaagaggaa ggtgtccaat ctcctgactg ttcaccagaa cctccctgcg ctgccagtag
     1681 atgccactag cgatgaggtc aggaaaaatc tcatggatat gtttagggat agacaggcgt
     1741 tttctgaaca cacctggaaa atgctgctta gcgtgtgccg atcctgggca gcctggtgta
     1801 agctgaacaa tcgcaaatgg ttccccgccg agccggagga cgtgcgcgat tacctgctgt
     1861 atctccaggc aagagggctg gctgtcaaga ctatccagca gcacttgggc caactgaata
     1921 tgctgcatcg acgcagcggg ctcccccggc ctagcgattc aaacgcagtc tcccttgtta
     1981 tgaggagaat tagaaaggaa aacgtagatg cgggtgagag ggctaagcag gctctcgctt
     2041 ttgagcggac tgatttcgac caggtcagat ccctgatgga gaacagcgat cggtgccagg
     2101 acatcaggaa cctcgcattt ctgggaattg catataacac acttctgcgc atagctgaga
     2161 tcgcccggat cagagtgaaa gacatcagtc gaacggacgg cggccggatg cttattcata
     2221 ttggacgcac aaagacattg gtcagcaccg ctggcgttga aaaggccttg tccctgggcg
     2281 taacgaagct ggtggaaaga tggatctcag tgtccggcgt ggctgacgac cctaataatt
     2341 acttgttctg tcgagtgaga aaaaacggag tcgccgcgcc ctctgccacc agccaattga
     2401 gtacacgggc ccttgaaggg atctttgagg caacccaccg actcatatac ggagccaagg
     2461 atgacagtgg ccagaggtat ctcgcctggt caggtcattc tgctagggtg ggggccgcac
     2521 gagacatggc gcgggcagga gtctccatac cagagattat gcaagctgga ggttggacaa
     2581 atgtgaacat cgttatgaac tatatccgca atcttgactc tgaaaccggg gccatggtga
     2641 gactgctcga agatggtgac taggaattcg atatctgctg gaagatggcg attagcatat
     2701 gggcattcac cgcgtgcctt actacgcgta ttcttaagca ataagcgaat ataacacggt
     2761 cgatctaaaa aggttagctg ggtgtgttag aagccctgca agacgacctg cgcattatta
     2821 ctcacggtac gacgcattat tactcacggt acgaggcatt caccgcgtgc cttactacgc
     2881 gtattcttaa gcaataagcg aatataacac ggtcgatcta aaaaggttag ctgggtgtgt
     2941 tagaagccct gcaagacgac ctgggcattc accgcgtgcc ttactacgcg tattcttaag
     3001 caataagcga atataacacg gtcgatctaa aaaggttagc tgggtgtgtt agaagccctg
     3061 caagacgacc tgggtaccgc attattactc acggtacgac gcattattac tcacggtacg
     3121 aggcattcac cgcgtgcctt actacgcgta ttcttaagca ataagcgaat ataacacggt
     3181 cgatctaaaa aggttagctg ggtgtgttag aagccctgca agacgacctg ggtaccaggt
     3241 aaagcggccg cgactctgat atcaagcttg cggccgcttc gagcagacat gataagatac
     3301 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3361 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3421 aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt tttttaaagc
     3481 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc atggctacgt
     3541 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc
     3601 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     3661 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagcctta attaacctaa
     3721 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3781 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3841 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     3901 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3961 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4021 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4081 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4141 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4201 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     4261 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4321 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4381 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4441 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4501 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4561 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4621 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4681 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4741 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4801 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4861 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4921 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4981 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5041 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5101 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     5161 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     5221 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     5281 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5341 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5401 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     5461 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5521 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5581 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5641 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5701 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5761 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5821 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5881 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5941 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6001 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6061 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6121 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6181 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6241 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6301 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6361 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
