LOCUS       sequence-430684-        7243 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 7243)
  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 7243)
  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-GeneTRAP
FEATURES             Location/Qualifiers
     source          1..7243
                     /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..1232
                     /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(670..688)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     938..957
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1274..1395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     intron          2237..2369
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2352..2371
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        2414..2432
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2453..2473
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4132..4253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   4415..4544
                     /label=AAV2 ITR
     primer_bind     complement(4563..4580)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4563..4579)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4572..4594)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4721..5176
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4808..4827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5018..5039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5193..5212)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5312..5334
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5372..5390)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5458..5562
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5563..5631,5632..6423)
                     /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      6594..7182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7083..7102
                     /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 gacggatgtg agcaagggcg
      541 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
      601 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
      661 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
      721 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
      781 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
      841 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
      901 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
      961 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     1021 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     1081 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     1141 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     1201 actccaccgg cggcatggac gagctgtaca agtgctgcat catcctgtaa gcggccgctt
     1261 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     1321 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     1381 tgcaataaac aagttggccg gggcggcagg cgggacgggt cgactctaga ctgcagaggg
     1441 agatctaaca tatcctggtg tggagtaggg gacgctgctc tgacagaggc tcgggggcct
     1501 gagctggctc tgtgagctgg ggaggaggca gacagccagg ccttgtctgc aagcagacct
     1561 ggcagcattg ggctggccgc cccccagggc ctcctcttca tgcccagtga atgactcacc
     1621 ttggcacaga cacaatgttc ggggtgggca cagtgcctgc ttcccgccgc accccagccc
     1681 ccctcaaatg ccttccgaga agcccattga gcagggggct tgcattgcac cccagcctga
     1741 cagcctggca tcttgggata aaagcagcac agccccctag gggctgccct tgctgtgtgg
     1801 cgccaccggc ggtggagaac aaggctctat tcagcctgtg cccaggaaag gggatcaggg
     1861 gatgcccagg catggacagt gggtggcagg gggggagagg agggctgtct gcttcccaga
     1921 agtccaagga cacaaatggg tgaggggaga gctctcccca tagctgggct gcggcccaac
     1981 cccaccccct caggctatgc cagggggtgt tgccaggggc acccgggcat cgccagtcta
     2041 gcccactcct tcataaagcc ctcgcatccc aggagcgagc agagccagag caggttggag
     2101 aggagacgca tcacctccgc tgctcgcaag ctttattgcg gtagtttatc acagttaaat
     2161 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
     2221 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
     2281 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
     2341 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     2401 aggctagagt acttaatacg actcactata ggctagcggt accgccacca tgcccaagaa
     2461 gaagaggaag gtgtccaatc tcctgactgt tcaccagaac ctccctgcgc tgccagtaga
     2521 tgccactagc gatgaggtca ggaaaaatct catggatatg tttagggata gacaggcgtt
     2581 ttctgaacac acctggaaaa tgctgcttag cgtgtgccga tcctgggcag cctggtgtaa
     2641 gctgaacaat cgcaaatggt tccccgccga gccggaggac gtgcgcgatt acctgctgta
     2701 tctccaggca agagggctgg ctgtcaagac tatccagcag cacttgggcc aactgaatat
     2761 gctgcatcga cgcagcgggc tcccccggcc tagcgattca aacgcagtct cccttgttat
     2821 gaggagaatt agaaaggaaa acgtagatgc gggtgagagg gctaagcagg ctctcgcttt
     2881 tgagcggact gatttcgacc aggtcagatc cctgatggag aacagcgatc ggtgccagga
     2941 catcaggaac ctcgcatttc tgggaattgc atataacaca cttctgcgca tagctgagat
     3001 cgcccggatc agagtgaaag acatcagtcg aacggacggc ggccggatgc ttattcatat
     3061 tggacgcaca aagacattgg tcagcaccgc tggcgttgaa aaggccttgt ccctgggcgt
     3121 aacgaagctg gtggaaagat ggatctcagt gtccggcgtg gctgacgacc ctaataatta
     3181 cttgttctgt cgagtgagaa aaaacggagt cgccgcgccc tctgccacca gccaattgag
     3241 tacacgggcc cttgaaggga tctttgaggc aacccaccga ctcatatacg gagccaagga
     3301 tgacagtggc cagaggtatc tcgcctggtc aggtcattct gctagggtgg gggccgcacg
     3361 agacatggcg cgggcaggag tctccatacc agagattatg caagctggag gttggacaaa
     3421 tgtgaacatc gttatgaact atatccgcaa tcttgactct gaaaccgggg ccatggtgag
     3481 actgctcgaa gatggtgact aggaattcga tatctgctgg aagatggcga ttagcatatg
     3541 ggcattcacc gcgtgcctta ctacgcgtat tcttaagcaa taagcgaata taacacggtc
     3601 gatctaaaaa ggttagctgg gtgtgttaga agccctgcaa gacgacctgc gcattattac
     3661 tcacggtacg acgcattatt actcacggta cgaggcattc accgcgtgcc ttactacgcg
     3721 tattcttaag caataagcga atataacacg gtcgatctaa aaaggttagc tgggtgtgtt
     3781 agaagccctg caagacgacc tgggcattca ccgcgtgcct tactacgcgt attcttaagc
     3841 aataagcgaa tataacacgg tcgatctaaa aaggttagct gggtgtgtta gaagccctgc
     3901 aagacgacct gggtaccgca ttattactca cggtacgacg cattattact cacggtacga
     3961 ggcattcacc gcgtgcctta ctacgcgtat tcttaagcaa taagcgaata taacacggtc
     4021 gatctaaaaa ggttagctgg gtgtgttaga agccctgcaa gacgacctgg gtaccaggta
     4081 aagcggccgc gactctgata tcaagcttgc ggccgcttcg agcagacatg ataagataca
     4141 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4201 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4261 acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt ttttaaagca
     4321 agtaaaacct ctacaaatgt ggtaaaatcg ataaggatct tcctagagca tggctacgta
     4381 gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc
     4441 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4501 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagccttaa ttaacctaat
     4561 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4621 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4681 cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     4741 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4801 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4861 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4921 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4981 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5041 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5101 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5161 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5221 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5281 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5341 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5401 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5461 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5521 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5581 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5641 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5701 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5761 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5821 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5881 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5941 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6001 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6061 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6121 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6181 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6241 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6301 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6361 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6421 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6481 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6541 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6601 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6661 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6721 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6781 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6841 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6901 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6961 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7021 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7081 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     7141 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     7201 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
