LOCUS       sequence-200933-        5825 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector including gRNA expression cassette and mEGFP knock-in 
            donor targeting mouse camk2a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5825)
  AUTHORS   Nishiyama J, Mikuni T, Yasuda R
  TITLE     Virus-Mediated Genome Editing via Homology-Directed Repair in 
            Mitotic and Postmitotic Cells in Mammalian Brain.
  JOURNAL   Neuron. 2017 Oct 18. pii: S0896-6273(17)30933-9. doi: 
            10.1016/j.neuron.2017.10.004.
  PUBMED    29056297
REFERENCE   2  (bases 1 to 5825)
  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: "Neuron. 
            2017 Oct 18. pii: S0896-6273(17)30933-9. doi: 
            10.1016/j.neuron.2017.10.004."
COMMENT     Sequence Label: pAAV-HDR-mEGFP-camk2a
FEATURES             Location/Qualifiers
     source          1..5825
                     /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             join(1441..1443,1444..1446,1447..1449,1450..2160)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MSVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1489..1510)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1750..1769)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2097..2118
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   3088..3228
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3088..3217
                     /label=AAV2 ITR
     rep_origin      3303..3758
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3390..3409)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3600..3621
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3775..3794)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3894..3916
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3954..3972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4040..4144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4145..4213,4214..5005)
                     /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(4363..4382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5176..5764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5665..5684
                     /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 aaacaccgct gcctgcccag tgccaggagt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt gaattcgctg tggcagaggc ctcaggaggg gccctgctgc
      541 tcagtggtga cagatagggg tgagaaagca gacagagtca ttccgtcagc attctgggtc
      601 tgtttggtac ttcttctcac gataaggtgg cggtgtgata tgcacaatgg ctaaaaagca
      661 gggagagctg gaaagaaaca aggacagaga cagaggccaa gtcaaccaga ccaattccca
      721 gaggaagcaa agaaaccatt acagagacta caagggggaa gggaaggaga gatgaattag
      781 cttcccctgt aaaccttaga acccagctgt tgccagggca acggggcaat acctgtctct
      841 tcagaggaga tgaagttgcc agggtaacta catcctgtct ttctcaagga ccatcccaga
      901 atgtggcacc cactagccgt taccatagca actgcctctt tgccccactt aatcccatcc
      961 cgtctgttaa aagggcccta tagttggagg tgggggaggt aggaagagcg atgatcactt
     1021 gtggactaag tttgttcgca tccccttctc caaccccctc agtacatcac cctgggggaa
     1081 cagggtccac ttgctcctgg gcccacacag tcctgcagta ttgtgtatat aaggccaggg
     1141 caaagaggag caggttttaa agtgaaaggc aggcaggtgt tggggaggca gttaccgggg
     1201 caacgggaac agggcgtttc ggaggtggtt gccatgggga cctggatgct gacgaaggct
     1261 cgcgaggctg tgagcagcca cagtgccctg ctcagaagcc ccaagctcgt cagtcaagcc
     1321 ggttctccgt ttgcactcag gagcacgggc aggcgagtgg cccctagttc tgggggcagc
     1381 gcttcagcat cccagcccta gttcccagcc taaagcctcg cctgcctgcc cagtgccagg
     1441 atgtccgtga gcaagggcga ggagctcttc accggggtgg tgcccatcct ggtcgagctg
     1501 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1561 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1621 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1681 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1741 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1801 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1861 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1921 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1981 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2041 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     2101 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2161 gctaccatca cctgcacccg attcacagaa gagtaccagc tctttgagga actgggaaag
     2221 tgagtcacac ctcagggagt cctagagagc atggggatca gtctccttgg gcagggggag
     2281 ggggcgtgtg cctatgtggc cagagctgtc ccgtgcatgc agagggaggt gtgcagcatg
     2341 atttggaagc actgtgtatg catagggtgt ccagagttag gcgctgacac atttatgagt
     2401 ccagtcagcc tggtgtgtgt gtgtgtgtgt gtgtgtgtga tggaaaggaa acagattggt
     2461 ggtgtatgca cacacctatg catataaata cacatgacct gggttcacaa agatatctag
     2521 aaacttgggg tttgaaaggg attttcccac ttggctggat ctgagctgga atctattttt
     2581 gtagttgtac ttagaaacct ccaatgacaa agaaagtttg ccaccccctg acctggagat
     2641 cttgtcttta gacactgtgc agagtggcta gctttctcct agcacatccc ctccggcttc
     2701 cccagatacc ttccgggagc aacagcttca gtacagaagg agttaatata aagcaatcac
     2761 agtgttggga ggaggggaag gacagtcagc cagccggggg gtgccactgt ggcactgact
     2821 atacaaaaca actgtggtcc tttggcaaag ttgcccatcg tgttatatct caggaggctg
     2881 agaggagaaa ggttccctcc agaagagcag caggctacag atataagaga aggtttctag
     2941 atgttcccag ctaggttatg gggtcctggg ttggatcagg agttggggga gcagtttggc
     3001 agagtgcccc acggatgcat acgtgctcct accagtcctg tttggttcct gctgtagcgc
     3061 agtaacatga gacgtcgacg cggccgcagg aacccctagt gatggagttg gccactccct
     3121 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     3181 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     3241 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     3301 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3361 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3421 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3481 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     3541 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3601 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     3661 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     3721 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     3781 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3841 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3901 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3961 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     4021 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4081 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4141 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     4201 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     4261 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     4321 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     4381 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     4441 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     4501 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4561 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4621 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4681 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4741 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4801 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4861 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4921 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4981 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     5041 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     5101 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     5161 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5221 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5281 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5341 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5401 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5461 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5521 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5581 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5641 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5701 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5761 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5821 catgt
//
