LOCUS       Exported                7438 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AAV vector for expression of SpyCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-CMV-SpyCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7438)
  AUTHORS   Bubeck F, Hoffmann MD, Harteveld Z, Aschenbrenner S, Bietz A, 
            Waldhauer MC, Borner K, Fakhiri J, Schmelas C, Dietz L, Grimm D, 
            Correia BE, Eils R, Niopek D
  TITLE     Engineered anti-CRISPR proteins for optogenetic control of 
            CRISPR-Cas9.
  JOURNAL   Nat Methods. 2018 Oct 30. pii: 10.1038/s41592-018-0178-9. doi: 
            10.1038/s41592-018-0178-9.
  PUBMED    30377362
REFERENCE   2  (bases 1 to 7438)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7438
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   530..659
                     /label=AAV2 ITR
     promoter        779..878
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     829..849
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      951..960
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             960..983
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             990..1010
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1035..5135
                     /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             5136..5183
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     repeat_region   5325..5454
                     /label=AAV2 ITR
     primer_bind     complement(5520..5537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5691..6279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5771..5790)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6450..7310)
                     /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     7073..7092
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7311..7415)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tgcgcgctcg
      541 ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc
      601 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt
      661 gtagttaatg attaacccgc catgctactt atctacgtag ccatgctcta gattaattaa
      721 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      781 aaaatcaacg ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg
      841 gtaggcgtgt acggtgggag gtctatataa gcagagctcg tttagtgaac cgtcagaatt
      901 ctcgagtgat cgaaagagcc tgctaaagca aaaaagaagt caccgctagc gccaccatgg
      961 attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg
     1021 gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg
     1081 gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca
     1141 acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg
     1201 aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga
     1261 accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct
     1321 tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc
     1381 ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc
     1441 acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg
     1501 ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg
     1561 acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg
     1621 aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga
     1681 gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc
     1741 tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg
     1801 acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca
     1861 acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt
     1921 ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc
     1981 tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag
     2041 ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga
     2101 acggctacgc cggctacatt gacggcggag ccagccagga agagttctac aagttcatca
     2161 agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg
     2221 acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg
     2281 gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc
     2341 gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca
     2401 ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga
     2461 acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca
     2521 acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt
     2581 acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc
     2641 ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc
     2701 ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact
     2761 ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc
     2821 tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg
     2881 aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga
     2941 aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca
     3001 ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca
     3061 agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga
     3121 tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg
     3181 gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca
     3241 tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg
     3301 agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca
     3361 gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga
     3421 aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc
     3481 agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg
     3541 atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc
     3601 tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga
     3661 agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt
     3721 tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca
     3781 tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact
     3841 cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca
     3901 ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg
     3961 agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc
     4021 tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg
     4081 acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact
     4141 tcttctacag caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga
     4201 tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg
     4261 gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa
     4321 agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg
     4381 ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc
     4441 ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac
     4501 tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga
     4561 atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca
     4621 agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg
     4681 ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt
     4741 acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc
     4801 tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct
     4861 ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc
     4921 accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca
     4981 atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca
     5041 ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg
     5101 agacacggat cgacctgtct cagctgggag gcgacaagcg tcctgctgct actaagaaag
     5161 ctggtcaagc taagaaaaag aaatgaatcg attagcaata aaggatcgtt tattttcatt
     5221 ggaagcgtgt gttggttttt tgatcaggcg cgggtaccag gtcgcggccg ctctagagca
     5281 tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat
     5341 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     5401 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcat
     5461 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5521 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5581 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5641 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5701 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5761 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5821 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5881 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5941 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6001 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6061 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6121 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6181 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6241 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6301 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6361 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6421 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6481 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6541 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6601 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6661 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6721 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6781 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6841 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6901 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6961 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7021 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7081 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7141 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7201 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7261 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7321 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7381 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
