LOCUS       Exported                5981 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector including gRNA expression cassette and mEGFP knock-in 
            donor targeting mouse Beta Actin.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-HDR-mEGFP-Actin
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5981)
  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 5981)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5981
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        149..389
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     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             1510..2229
                     /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(1558..1579)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1819..1838)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2166..2187
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   3244..3384
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3244..3373
                     /label=AAV2 ITR
     rep_origin      3459..3914
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3546..3565)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3756..3777
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3931..3950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4050..4072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4110..4128)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4196..4300
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4301..5161
                     /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(4519..4538)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5332..5920
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5821..5840
                     /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 aaacaccgtg tgtcttgata gttcgccagt
      421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
      481 caccgagtcg gtgctttttt tgaattctgc agctccttcg ttgccggtcc acacccgcca
      541 ccaggtaagc agggacgccg ggcccagcgg gccttcgctc tctcgtggct agtacctcac
      601 tgcagggtcc tgaggatcac tcagaacgga caccatgggc gggtggaggg tggtgccggg
      661 ccgcggagcg gacactggca cagccaactt tacgcctagc gtgtagactc tttgcagcca
      721 cattcccgcg gtgtagacac tcgtgggccc gctcccgctc ggtgcgtggg gcttggggac
      781 acactagggt cgcggtgtgg gcatttgatg agccggtgcg gcttgcgggt gttaaaagcc
      841 gtattaggtc catcttgaga gtacacagta ttgggaacca gacgctacga tcacgcctca
      901 atggcctctg ggtctttgtc caaaccggtt tgcctattcg gcttgccggg cgggcgggcg
      961 ggcgggcggg cgcggcaggg ccggctcggc cgggtggggg ctgggatgcc actgcgcgtg
     1021 cgctctctat cactgggcat cgaggcgcgt gtgcgctagg gagggagctc ttcctctccc
     1081 cctcttccta gttagctgcg cgtgcgtatt gaggctggga gcgcggctgc ccggggttgg
     1141 gcgagggcgg ggccgttgtc cggaaggggc ggggtcacag tggcacgggc gccttgtttg
     1201 cgcttcctgc tgggtgtggt cgcctcccgc gcgcgcacaa gccgcccgtc ggcgcagtgt
     1261 aggcggagct tgcgcccgtt tggggagggg gcggaggtct ggcttcctgc cctaggtccg
     1321 cctccgggcc agcgtttgcc ttttatggta ataatgcggc cggtctgcgc ttcctttgtc
     1381 ccctgagctt gggcgcgcgc cccctggcgg ctcgagcccg cggcttgccg gaagtgggca
     1441 gggcggcagc ggctgctctt ggcggccccg aggtgactat agccttcttt tgtgtcttga
     1501 tagttcgcca tgcatgtgag caagggcgag gagctcttca ccggggtggt gcccatcctg
     1561 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1621 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1681 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     1741 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1801 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1861 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1921 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1981 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     2041 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2101 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     2161 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     2221 ctgtacaagg atgacgatat cgctgcgctg gtcgtcgaca acggctccgg catgtgcaaa
     2281 gccggcttcg cgggcgacga tgctccccgg gctgtattcc cctccatcgt gggccgccct
     2341 aggcaccagg taagtgacct gttactttgg gagtggcaag cctggggttt tcttggggat
     2401 cgatgccggt gctaagaagg ctgttccctt ccacagggtg tgatggtggg aatgggtcag
     2461 aaggactcct atgtgggtga cgaggcccag agcaagagag gtatcctgac cctgaagtac
     2521 cccattgaac atggcattgt taccaactgg gacgacatgg agaagatctg gcaccacacc
     2581 ttctacaatg agctgcgtgt ggcccctgag gagcaccctg tgctgctcac cgaggccccc
     2641 ctgaacccta aggccaaccg tgaaaagatg acccaggtca gtatcccggg taacccttct
     2701 ctttggccag cttctcagcc acgccctttc tcaattgtct ttcttctgcc gttctcccat
     2761 aggactccct tctatgagct tagtctccct tggatctttg cagtttctgc tctttcccag
     2821 acgaggtctt tttttctctc aattgccttt ctgactaggt gtttaaaccc tacagtgctg
     2881 tgggtttagg tactaacaat ggctcgtgtg acaaagctaa tgaggctggt gataagtggc
     2941 cttggattgt gtattgagta gatgcacagt aggtctaagt ggagcccctg tcctgagact
     3001 cccagcacac tgaacttagc tgtgttcttg cactccttgc atgtctcaga tctatccata
     3061 cagtttcacc tgccctgagt gtttcttgtg gctttctgaa cttgacaaca ttatttattt
     3121 ttctctacag atcatgtttg agaccttcaa caccccagcc atgtacgtag ccatccaggc
     3181 tgtgctgtcc ctgtatgcct ctggtcgtac cacaggcatt gtgatggacg gatccgcggc
     3241 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     3301 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     3361 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     3421 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     3481 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     3541 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3601 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3661 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3721 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3781 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     3841 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3901 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3961 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4021 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4081 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4141 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4201 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4261 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4321 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4381 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4441 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4501 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4561 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4621 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4681 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4741 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4801 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4861 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4921 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4981 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     5041 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5101 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5161 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5221 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5281 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5341 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5401 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5461 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5521 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5581 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     5641 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     5701 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     5761 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     5821 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     5881 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     5941 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
