LOCUS       sequence-388474-        5816 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 5816)
  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 5816)
  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: pGEARBOCS-v0-Vamp2-TagIN-HA
FEATURES             Location/Qualifiers
     source          1..5816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(435..455)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(476..494)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(537..556)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          539..671
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             complement(1515..1541)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_RNA        complement(1579..1654)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1676..1924)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1734..1753)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1904..1924)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   1939..2068
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2130..2718)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2210..2229)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2889..3680,3681..3749))
                     /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"
     promoter        complement(3750..3854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3922..3940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3978..4000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4100..4119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4136..4591)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4273..4294)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4485..4504
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4718..4740
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4732..4749
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4733..4749
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   4768..4897
                     /label=AAV2 ITR
     polyA_signal    5059..5180
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5096..5115)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5150..5169
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 ctgacctggt cgaaatcagt ccgctcaaaa gcgagagcct gcttagccct ctcacccgca
       61 tctacgtttt cctttctaat tctcctcata acaagggaga ctgcgtttga atcgctaggc
      121 cgggggagcc cgctgcgtcg atgcagcata ttcagttggc ccaagtgctg ctggatagtc
      181 ttgacagcca gccctcttgc ctggagatac agcaggtaat cgcgcacgtc ctccggctcg
      241 gcggggaacc atttgcgatt gttcagctta caccaggctg cccaggatcg gcacacgcta
      301 agcagcattt tccaggtgtg ttcagaaaac gcctgtctat ccctaaacat atccatgaga
      361 tttttcctga cctcatcgct agtggcatct actggcagcg cagggaggtt ctggtgaaca
      421 gtcaggagat tggacacctt cctcttcttc ttgggcatgg tggcggtacc gctagcctat
      481 agtgagtcgt attaagtact ctagccttaa gagctgtaat tgaactggga gtggacacct
      541 gtggagagaa aggcaaagtg gatgtcagta agaccaatag gtgcctatca gaaacgcaag
      601 agtcttctct gtctcgacaa gcccagtttc tattggtctc cttaaacctg tcttgtaacc
      661 ttgatactta cctgcccagt gcctcacgac caacttctgc agcttaagtt cgagactgtt
      721 gtgtcagaag cactgactgc gttagcaatt taactgtgat aaactaccgc aataaagctt
      781 gcgagcagcg gaggtgatgc gtctcctctc caacctgctc tggctctgct cgctcctggg
      841 atgcgagggc tttatgaagg agtgggctag actggcgatg cccgggtgcc cctggcaaca
      901 ccccctggca tagcctgagg gggtggggtt gggccgcagc ccagctatgg ggagagctct
      961 cccctcaccc atttgtgtcc ttggacttct gggaagcaga cagccctcct ctccccccct
     1021 gccacccact gtccatgcct gggcatcccc tgatcccctt tcctgggcac aggctgaata
     1081 gagccttgtt ctccaccgcc ggtggcgcca cacagcaagg gcagccccta gggggctgtg
     1141 ctgcttttat cccaagatgc caggctgtca ggctggggtg caatgcaagc cccctgctca
     1201 atgggcttct cggaaggcat ttgagggggg ctggggtgcg gcgggaagca ggcactgtgc
     1261 ccaccccgaa cattgtgtct gtgccaaggt gagtcattca ctgggcatga agaggaggcc
     1321 ctggggggcg gccagcccaa tgctgccagg tctgcttgca gacaaggcct ggctgtctgc
     1381 ctcctcccca gctcacagag ccagctcagg cccccgagcc tctgtcagag cagcgtcccc
     1441 tactccacac caggatatgt tagatctccc tctgcagtct agagtcgacc cgtcccgcct
     1501 gccgccccgg ccgtagcgta gtctggaacg tcgtaagggt aatccgtccc gcctgccgcc
     1561 ccggccggat ccaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag
     1621 ccttatttta acttgctatt tctagctcta aaactcccgc ctgccgcccc ggcccggtgt
     1681 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     1741 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     1801 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa
     1861 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     1921 cctcacgcgt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     1981 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc ggcctcagtg
     2041 agcgagcgag cgcgcagctg cctgcaggac atgtgagcaa aaggccagca aaaggccagg
     2101 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2161 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2221 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2281 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2341 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2401 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2461 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2521 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2581 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2641 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2701 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2761 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2821 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2881 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2941 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3001 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3061 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3121 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3181 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3241 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3301 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3361 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3421 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3481 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3541 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3601 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3661 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3721 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3781 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3841 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     3901 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     3961 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4021 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4081 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccataaaatt
     4141 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4201 aaccaataga ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagatagag
     4261 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4321 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     4381 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4441 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4501 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4561 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     4621 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     4681 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     4741 acggccagtg aattaggtta attaaggctg cgcgctcgct cgctcactga ggccgcccgg
     4801 gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
     4861 agagagggag tggccaactc catcactagg ggttccttgt agttaatgat taacccgcca
     4921 tgctacttat ctacgtagcc atgctctagg aagatcctta tcgattttac cacatttgta
     4981 gaggttttac ttgctttaaa aaacctccca catctccccc tgaacctgaa acataaaatg
     5041 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5101 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5161 aaactcatca atgtatctta tcatgtctgc tcgaagcggc cgcaagcttg atatcgaatt
     5221 cctagtcacc atcttcgagc agtctcacca tggccccggt ttcagagtca agattgcgga
     5281 tatagttcat aacgatgttc acatttgtcc aacctccagc ttgcataatc tctggtatgg
     5341 agactcctgc ccgcgccatg tctcgtgcgg cccccaccct agcagaatga cctgaccagg
     5401 cgagatacct ctggccactg tcatccttgg ctccgtatat gagtcggtgg gttgcctcaa
     5461 agatcccttc aagggcccgt gtactcaatt ggctggtggc agagggcgcg gcgactccgt
     5521 tttttctcac tcgacagaac aagtaattat tagggtcgtc agccacgccg gacactgaga
     5581 tccatctttc caccagcttc gttacgccca gggacaaggc cttttcaacg ccagcggtgc
     5641 tgaccaatgt ctttgtgcgt ccaatatgaa taagcatccg gccgccgtcc gttcgactga
     5701 tgtctttcac tctgatccgg gcgatctcag ctatgcgcag aagtgtgtta tatgcaattc
     5761 ccagaaatgc gaggttcctg atgtcctggc accgatcgct gttctccatc agggat
//
