LOCUS       Exported                7700 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses SpdCas9 from the nEF promoter in AAV backbone.
ACCESSION   .
VERSION     .
KEYWORDS    AAV-nEF-dCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7700)
  AUTHORS   Liao HK, Hatanaka F, Araoka T, Reddy P, Wu MZ, Sui Y, Yamauchi T, 
            Sakurai M, O'Keefe DD, Nunez-Delicado E, Guillen P, Campistol JM, Wu
            CJ, Lu LF, Esteban CR, Izpisua Belmonte JC
  TITLE     In Vivo Target Gene Activation via CRISPR/Cas9-Mediated 
            Trans-epigenetic Modulation.
  JOURNAL   Cell. 2017 Dec 14;171(7):1495-1507.e15. doi: 
            10.1016/j.cell.2017.10.025. Epub 2017 Dec 7.
  PUBMED    29224783
REFERENCE   2  (bases 1 to 7700)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7700
                     /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        175..386
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             399..667
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             704..730
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             734..754
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             764..4864
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4868..4888
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4907..4955
                     /note="synthetic polyadenylation signal"
     repeat_region   4963..5103
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4963..5092
                     /label=AAV2 ITR
     rep_origin      5178..5633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5265..5284)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5475..5496
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5650..5669)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5769..5791
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5829..5847)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5915..6019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6020..6880
                     /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(6238..6257)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7051..7639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7540..7559
                     /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 gaaaggatct gcgatcgctc cggtgcccgt cagtgggcag
      181 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaacgggtg
      241 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      301 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      361 gcaacgggtt tgccgccaga acacagctga agcttcgagg ggctcgcatc tctccttcac
      421 gcgcccgccg ccctacctga ggccgccatc cacgccggtt gagtcgcgtt ctgccgcctc
      481 ccgcctgtgg tgcctcctga actgcgtccg ccgtctaggt aagtttaaag ctcaggtcga
      541 gaccgggcct ttgtccggcg ctcccttgga gcctacctag actcagccgg ctctccacgc
      601 tttgcctgac cctgcttgct caactctacg tctttgtttc gttttctgtt ctgcgccgtt
      661 acagatccaa gctgtgaccg gcgcctacac cggtgccacc atgtacccat acgatgttcc
      721 agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga agtactccat
      781 tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc attacggacg agtacaaggt
      841 gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct
      901 cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac
      961 agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag
     1021 taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt
     1081 ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc
     1141 gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga
     1201 taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca
     1261 cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca
     1321 actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga
     1381 cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc
     1441 acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg
     1501 gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag
     1561 caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc
     1621 agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg
     1681 agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga
     1741 gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta
     1801 caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc
     1861 aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga
     1921 ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa
     1981 tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga
     2041 ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg
     2101 gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg
     2161 caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc
     2221 tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt
     2281 gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt
     2341 caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc
     2401 tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga
     2461 ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt
     2521 caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct
     2581 ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga
     2641 agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt
     2701 catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat
     2761 caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg
     2821 atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga
     2881 catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca tcgctaatct
     2941 tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact
     3001 cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa
     3061 ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg
     3121 tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca
     3181 gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga
     3241 actggacatc aatcggctct ccgactacga cgtggctgct atcgtgcccc agtcttttct
     3301 caaagatgat tctattgata ataaagtgtt gacaagatcc gataaagcta gagggaagag
     3361 tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct
     3421 gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg
     3481 cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat
     3541 caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga
     3601 caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct cagatttcag
     3661 aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc
     3721 ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga
     3781 atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca
     3841 ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa
     3901 gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg
     3961 agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct
     4021 gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa
     4081 ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga
     4141 ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc
     4201 caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac
     4261 aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata
     4321 taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga
     4381 aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc
     4441 actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg
     4501 gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga
     4561 tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct
     4621 cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga
     4681 aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt
     4741 cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact
     4801 gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg
     4861 agacagcccc aagaagaaga gaaaggtgga ggccagctaa gaattcaata aaagatcttt
     4921 attttcatta gatctgtgtg ttggtttttt gtgtgcggcc gcaggaaccc ctagtgatgg
     4981 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     5041 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     5101 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     5161 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5221 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     5281 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5341 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5401 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5461 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5521 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5581 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5641 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5701 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5761 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5821 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5881 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5941 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     6001 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     6061 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6121 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6181 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6241 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6301 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6361 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6421 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6481 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6541 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6601 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6661 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6721 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6781 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6841 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6901 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6961 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     7021 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     7081 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7141 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7201 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7261 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7321 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7381 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7441 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7501 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7561 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     7621 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     7681 ctggcctttt gctcacatgt
//
