LOCUS       sequence-227073-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector including gRNA expression cassette and mEGFP knock-in 
            donor targeting mouse Beta Actin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  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 5992)
  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-Actin
FEATURES             Location/Qualifiers
     source          1..5992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        430..505
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(1521..1523,1524..1526,1527..1529,1530..2240)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MHVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD
                     DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGI
                     KVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVL
                     LEFVTAAGITLGMDELYK"
     primer_bind     complement(1569..1590)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1830..1849)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2177..2198
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   3255..3395
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3255..3384
                     /label=AAV2 ITR
     rep_origin      3470..3925
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3557..3576)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3767..3788
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3942..3961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4061..4083
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4121..4139)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4207..4311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4312..4380,4381..5172)
                     /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(4530..4549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5343..5931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5832..5851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agaaagcttg agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgt gtgtcttgat
      421 agttcgccag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact
      481 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaattctg cagctccttc gttgccggtc
      541 cacacccgcc accaggtaag cagggacgcc gggcccagcg ggccttcgct ctctcgtggc
      601 tagtacctca ctgcagggtc ctgaggatca ctcagaacgg acaccatggg cgggtggagg
      661 gtggtgccgg gccgcggagc ggacactggc acagccaact ttacgcctag cgtgtagact
      721 ctttgcagcc acattcccgc ggtgtagaca ctcgtgggcc cgctcccgct cggtgcgtgg
      781 ggcttgggga cacactaggg tcgcggtgtg ggcatttgat gagccggtgc ggcttgcggg
      841 tgttaaaagc cgtattaggt ccatcttgag agtacacagt attgggaacc agacgctacg
      901 atcacgcctc aatggcctct gggtctttgt ccaaaccggt ttgcctattc ggcttgccgg
      961 gcgggcgggc gggcgggcgg gcgcggcagg gccggctcgg ccgggtgggg gctgggatgc
     1021 cactgcgcgt gcgctctcta tcactgggca tcgaggcgcg tgtgcgctag ggagggagct
     1081 cttcctctcc ccctcttcct agttagctgc gcgtgcgtat tgaggctggg agcgcggctg
     1141 cccggggttg ggcgagggcg gggccgttgt ccggaagggg cggggtcaca gtggcacggg
     1201 cgccttgttt gcgcttcctg ctgggtgtgg tcgcctcccg cgcgcgcaca agccgcccgt
     1261 cggcgcagtg taggcggagc ttgcgcccgt ttggggaggg ggcggaggtc tggcttcctg
     1321 ccctaggtcc gcctccgggc cagcgtttgc cttttatggt aataatgcgg ccggtctgcg
     1381 cttcctttgt cccctgagct tgggcgcgcg ccccctggcg gctcgagccc gcggcttgcc
     1441 ggaagtgggc agggcggcag cggctgctct tggcggcccc gaggtgacta tagccttctt
     1501 ttgtgtcttg atagttcgcc atgcatgtga gcaagggcga ggagctcttc accggggtgg
     1561 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1621 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1681 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     1741 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1801 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1861 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1921 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     1981 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2041 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2101 ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc aaagacccca
     2161 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2221 gcatggacga gctgtacaag gatgacgata tcgctgcgct ggtcgtcgac aacggctccg
     2281 gcatgtgcaa agccggcttc gcgggcgacg atgctccccg ggctgtattc ccctccatcg
     2341 tgggccgccc taggcaccag gtaagtgacc tgttactttg ggagtggcaa gcctggggtt
     2401 ttcttgggga tcgatgccgg tgctaagaag gctgttccct tccacagggt gtgatggtgg
     2461 gaatgggtca gaaggactcc tatgtgggtg acgaggccca gagcaagaga ggtatcctga
     2521 ccctgaagta ccccattgaa catggcattg ttaccaactg ggacgacatg gagaagatct
     2581 ggcaccacac cttctacaat gagctgcgtg tggcccctga ggagcaccct gtgctgctca
     2641 ccgaggcccc cctgaaccct aaggccaacc gtgaaaagat gacccaggtc agtatcccgg
     2701 gtaacccttc tctttggcca gcttctcagc cacgcccttt ctcaattgtc tttcttctgc
     2761 cgttctccca taggactccc ttctatgagc tgagtctccc ttggatcttt gcagtttctg
     2821 ctctttccca gacgaggtct ttttttctct caattgcctt tctgactagg tgtttaaacc
     2881 ctacagtgct gtgggtttag gtactaacaa tggctcgtgt gacaaagcta atgaggctgg
     2941 tgataagtgg ccttggagtg tgtattgagt agatgcacag taggtctaag tggagcccct
     3001 gtcctgagac tcccagcaca ctgaacttag ctgtgttctt gcactccttg catgtctcag
     3061 atctatccat acagtttcac ctgccctgag tgtttcttgt ggctttctga acttgacaac
     3121 attatttatt tttctctaca gatcatgttt gagaccttca acaccccagc catgtacgta
     3181 gccatccagg ctgtgctgtc cctgtatgcc tctggtcgta ccacaggcat tgtgatggac
     3241 ggatccgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3301 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     3361 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     3421 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3481 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3541 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3601 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3661 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     3721 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3781 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg
     3841 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3901 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     3961 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4021 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4081 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     4141 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4201 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4261 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4321 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4381 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4441 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4501 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4561 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4621 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4681 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4741 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4801 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4861 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4921 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4981 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     5041 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     5101 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     5161 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5221 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5281 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5341 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5401 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5461 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     5521 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5581 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5641 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5701 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5761 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5821 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5881 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5941 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
