LOCUS       Exported                7389 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pAAV-pMecp2-SpCas9-spA (AAV-SpCas9). AAV plasmid expressing Cas9 in 
            neurons under control of truncated mecp2 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    PX551
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7389)
  AUTHORS   Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur
            M, Zhang F
  TITLE     In vivo interrogation of gene function in the mammalian brain using 
            CRISPR-Cas9.
  JOURNAL   Nat Biotechnol. 2014 Oct 19. doi: 10.1038/nbt.3055.
  PUBMED    25326897
REFERENCE   2  (bases 1 to 7389)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7389
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        106..334
                     /label=Mecp2 promoter
                     /note="promoter for selective expression in neurons (Adachi
                     et al., 2005; Gray et al., 2011)"
     CDS             350..376
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             380..400
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             410..4510
                     /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             4514..4534
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4553..4601
                     /note="synthetic polyadenylation signal"
     repeat_region   4609..4749
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4609..4738
                     /label=AAV2 ITR
     rep_origin      4824..5279
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4911..4930)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5121..5142
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5296..5315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5415..5437
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5475..5493)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5561..5665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5666..6526
                     /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(5884..5903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6697..7285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7186..7205
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7347..87
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag
       61 tggccaactc catcactagg ggttcctgcg gcctctagaa agcttagctg aatggggtcc
      121 gcctcttttc cctgcctaaa cagacaggaa ctcctgccaa ttgagggcgt caccgctaag
      181 gctccgcccc agcctgggct ccacaaccaa tgaagggtaa tctcgacaaa gagcaagggg
      241 tggggcgcgg gcgcgcaggt gcagcagcac acaggctggt cgggagggcg gggcgcgacg
      301 tctgccgtgc ggggtcccgg catcggttgc gcgcaccggt gccaccatgt acccatacga
      361 tgttccagat tacgcttcgc cgaagaaaaa gcgcaaggtc gaagcgtccg acaagaagta
      421 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
      481 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
      541 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
      601 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
      661 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
      721 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
      781 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
      841 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
      901 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
      961 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     1021 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     1081 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     1141 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     1201 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     1261 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     1321 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     1381 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     1441 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     1501 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     1561 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     1621 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     1681 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     1741 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     1801 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     1861 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     1921 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     1981 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     2041 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     2101 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     2161 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     2221 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     2281 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     2341 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     2401 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     2461 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     2521 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     2581 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     2641 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     2701 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     2761 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     2821 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     2881 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     2941 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3001 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     3061 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     3121 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     3181 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     3241 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     3301 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     3361 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     3421 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     3481 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     3541 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     3601 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     3661 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     3721 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     3781 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     3841 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     3901 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     3961 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4021 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     4081 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     4141 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     4201 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     4261 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     4321 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     4381 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     4441 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     4501 gggaggcgac agccccaaga agaagagaaa ggtggaggcc agctaagaat tcaataaaag
     4561 atctttattt tcattagatc tgtgtgttgg ttttttgtgt gcggccgcag gaacccctag
     4621 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4681 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4741 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4801 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4861 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4921 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4981 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5041 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5101 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5161 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5221 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5281 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5341 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5401 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5461 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5521 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5581 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5641 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5701 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5761 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5821 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5881 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5941 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6001 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6061 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6121 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6181 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6241 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6301 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6361 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     6421 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6481 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6541 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6601 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6661 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6721 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6781 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6841 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6901 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6961 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7021 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7081 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7141 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7201 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7261 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7321 cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct cgctcactga
     7381 ggccgcccg
//
