LOCUS       Exported                7418 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector expressing Myc-tagged SpCas9 under the EFS promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-EFS-SpCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7418)
  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 7418)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7418
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..780
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        841..1052
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      1060..1069
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1069..1098
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1102..1122
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1132..5232
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5236..5256
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5275..5323
                     /note="synthetic polyadenylation signal"
     repeat_region   5331..5471
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5331..5460
                     /label=AAV2 ITR
     rep_origin      5546..6001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5633..5652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5843..5864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6018..6037)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6137..6159
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6197..6215)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6283..6387
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6388..7248
                     /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(6606..6625)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg
      721 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
      781 gcggcctcta gaaagcttag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
      841 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat
      901 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      961 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     1021 tttttcgcaa cgggtttgcc gccagaacac aggaccggtg ccaccatgga acaaaaactc
     1081 atctcagaag aggatctgtc gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag
     1141 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag
     1201 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag
     1261 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg
     1321 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag
     1381 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc
     1441 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac
     1501 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac
     1561 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc
     1621 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg
     1681 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc
     1741 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat
     1801 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg
     1861 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg
     1921 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac
     1981 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac
     2041 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga
     2101 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct
     2161 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac
     2221 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac
     2281 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc
     2341 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg
     2401 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg
     2461 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg
     2521 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag
     2581 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac
     2641 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg
     2701 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag
     2761 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg
     2821 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa
     2881 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag
     2941 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca
     3001 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac
     3061 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg
     3121 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag
     3181 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt
     3241 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt
     3301 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg
     3361 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc
     3421 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc
     3481 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc
     3541 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg
     3601 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag
     3661 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg
     3721 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg
     3781 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag
     3841 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc
     3901 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac
     3961 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc
     4021 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc
     4081 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg
     4141 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag
     4201 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac
     4261 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag
     4321 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg
     4381 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc
     4441 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag
     4501 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg
     4561 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg
     4621 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc
     4681 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc
     4741 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac
     4801 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag
     4861 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac
     4921 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac
     4981 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag
     5041 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc
     5101 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac
     5161 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag
     5221 ctgggaggcg acagccccaa gaagaagaga aaggtggagg ccagctaaga attcaataaa
     5281 agatctttat tttcattaga tctgtgtgtt ggttttttgt gtgcggccgc aggaacccct
     5341 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     5401 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     5461 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5521 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     5641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5761 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     5881 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5941 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     6001 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     6061 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6121 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6181 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     6241 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     6301 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6361 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6421 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6481 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6541 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6601 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6661 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6721 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6781 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6841 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6901 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6961 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     7021 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     7081 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     7141 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     7201 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     7261 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     7321 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7381 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
