LOCUS       sequence-388467-        6655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To KO and genetrap mouse VAMP2 simultaneously.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  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 6655)
  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-GeneTRAP
FEATURES             Location/Qualifiers
     source          1..6655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(423..443)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     promoter        complement(464..482)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(525..544)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          527..659
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     polyA_signal    1501..1622
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(1664..2368)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(1939..1958)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2208..2226
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_RNA        complement(2406..2481)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2503..2751)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2561..2580)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2731..2751)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2766..2895
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(2957..3545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3037..3056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3716..4507,4508..4576))
                     /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(4577..4681)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4749..4767
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4805..4827)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4927..4946
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(4963..5418)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5100..5121)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5312..5331
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5545..5567
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5559..5576
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5560..5576
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   5595..5724
                     /label=AAV2 ITR
     polyA_signal    5886..6007
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 aaatcagtcc gctcaaaagc gagagcctgc ttagccctct cacccgcatc tacgttttcc
       61 tttctaattc tcctcataac aagggagact gcgtttgaat cgctaggccg ggggagcccg
      121 ctgcgtcgat gcagcatatt cagttggccc aagtgctgct ggatagtctt gacagccagc
      181 cctcttgcct ggagatacag caggtaatcg cgcacgtcct ccggctcggc ggggaaccat
      241 ttgcgattgt tcagcttaca ccaggctgcc caggatcggc acacgctaag cagcattttc
      301 caggtgtgtt cagaaaacgc ctgtctatcc ctaaacatat ccatgagatt tttcctgacc
      361 tcatcgctag tggcatctac tggcagcgca gggaggttct ggtgaacagt caggagattg
      421 gacaccttcc tcttcttctt gggcatggtg gcggtaccgc tagcctatag tgagtcgtat
      481 taagtactct agccttaaga gctgtaattg aactgggagt ggacacctgt ggagagaaag
      541 gcaaagtgga tgtcagtaag accaataggt gcctatcaga aacgcaagag tcttctctgt
      601 ctcgacaagc ccagtttcta ttggtctcct taaacctgtc ttgtaacctt gatacttacc
      661 tgcccagtgc ctcacgacca acttctgcag cttaagttcg agactgttgt gtcagaagca
      721 ctgactgcgt tagcaattta actgtgataa actaccgcaa taaagcttgc gagcagcgga
      781 ggtgatgcgt ctcctctcca acctgctctg gctctgctcg ctcctgggat gcgagggctt
      841 tatgaaggag tgggctagac tggcgatgcc cgggtgcccc tggcaacacc ccctggcata
      901 gcctgagggg gtggggttgg gccgcagccc agctatgggg agagctctcc cctcacccat
      961 ttgtgtcctt ggacttctgg gaagcagaca gccctcctct ccccccctgc cacccactgt
     1021 ccatgcctgg gcatcccctg atcccctttc ctgggcacag gctgaataga gccttgttct
     1081 ccaccgccgg tggcgccaca cagcaagggc agcccctagg gggctgtgct gcttttatcc
     1141 caagatgcca ggctgtcagg ctggggtgca atgcaagccc cctgctcaat gggcttctcg
     1201 gaaggcattt gaggggggct ggggtgcggc gggaagcagg cactgtgccc accccgaaca
     1261 ttgtgtctgt gccaaggtga gtcattcact gggcatgaag aggaggccct ggggggcggc
     1321 cagcccaatg ctgccaggtc tgcttgcaga caaggcctgg ctgtctgcct cctccccagc
     1381 tcacagagcc agctcaggcc cccgagcctc tgtcagagca gcgtccccta ctccacacca
     1441 ggatatgtta gatctccctc tgcagtctag agtcgacccg tcccgcctgc cgccccggcc
     1501 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1561 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1621 tatcatgtct gctcgaagcg gccgcttaca ggatgatgca gcacttgtac agctcgtcca
     1681 tgccgccggt ggagtggcgg ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct
     1741 cgttgtggga ggtgatgtcc aacttgatgt tgacgttgta ggcgccgggc agctgcacgg
     1801 gcttcttggc cttgtaggtg gtcttgacct cagcgtcgta gtggccgccg tccttcagct
     1861 tcagcctctg cttgatctcg cccttcaggg cgccgtcctc ggggtacatc cgctcggagg
     1921 aggcctccca gcccatggtc ttcttctgca ttacggggcc gtcggagggg aagttggtgc
     1981 cgcgcagctt caccttgtag atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg
     2041 tcaccacgcc gccgtcctcg aagttcatca cgcgctccca cttgaagccc tcggggaagg
     2101 acagcttcaa gtagtcgggg atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca
     2161 tgaactgagg ggacaggatg tcccaggcga agggcagggg gccacccttg gtcaccttca
     2221 gcttggcggt ctgggtgccc tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact
     2281 cgtggccgtt cacggagccc tccatgtgca ccttgaagcg catgaactcc ttgatgatgg
     2341 ccatgttatc ctcctcgccc ttgctcacat ccgtcccgcc tgccgccccg gccggatcca
     2401 aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct tattttaact
     2461 tgctatttct agctctaaaa ctcccgcctg ccgccccggc ccggtgtttc gtcctttcca
     2521 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
     2581 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
     2641 cgtattttgt actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac
     2701 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct cacgcgtgcg
     2761 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     2821 tgaggccggg cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc gagcgagcgc
     2881 gcagctgcct gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2941 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3001 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3061 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3121 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3181 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3241 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3301 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3361 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     3421 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3481 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3541 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3601 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     3661 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     3721 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3781 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     3841 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     3901 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3961 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4021 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4081 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4141 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4201 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4261 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4321 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4381 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4441 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4501 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4561 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4621 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4681 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     4741 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     4801 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4861 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     4921 actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta aacgttaata
     4981 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caatagaccg
     5041 aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg agtgttgttc
     5101 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5161 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5221 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5281 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5341 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5401 cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5461 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     5521 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
     5581 taggttaatt aaggctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc
     5641 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     5701 ccaactccat cactaggggt tccttgtagt taatgattaa cccgccatgc tacttatcta
     5761 cgtagccatg ctctaggaag atccttatcg attttaccac atttgtagag gttttacttg
     5821 ctttaaaaaa cctcccacat ctccccctga acctgaaaca taaaatgaat gcaattgttg
     5881 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5941 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     6001 tatcttatca tgtctgctcg aagcggccgc aagcttgata tcgaattcct agtcaccatc
     6061 ttcgagcagt ctcaccatgg ccccggtttc agagtcaaga ttgcggatat agttcataac
     6121 gatgttcaca tttgtccaac ctccagcttg cataatctct ggtatggaga ctcctgcccg
     6181 cgccatgtct cgtgcggccc ccaccctagc agaatgacct gaccaggcga gatacctctg
     6241 gccactgtca tccttggctc cgtatatgag tcggtgggtt gcctcaaaga tcccttcaag
     6301 ggcccgtgta ctcaattggc tggtggcaga gggcgcggcg actccgtttt ttctcactcg
     6361 acagaacaag taattattag ggtcgtcagc cacgccggac actgagatcc atctttccac
     6421 cagcttcgtt acgcccaggg acaaggcctt ttcaacgcca gcggtgctga ccaatgtctt
     6481 tgtgcgtcca atatgaataa gcatccggcc gccgtccgtt cgactgatgt ctttcactct
     6541 gatccgggcg atctcagcta tgcgcagaag tgtgttatat gcaattccca gaaatgcgag
     6601 gttcctgatg tcctggcacc gatcgctgtt ctccatcagg gatctgacct ggtcg
//
