LOCUS       40924_47093        7090 bp DNA     circular SYN 13-MAY-2021
DEFINITION  A catalytically inactive SaCas9 (dSaCas9) with the D10A and N580A 
            mutations. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7090)
  AUTHORS   Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B,
            Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F
  TITLE     In vivo genome editing using Staphylococcus aureus Cas9.
  JOURNAL   Nature. 2015 Apr 1. doi: 10.1038/nature14299.
  PUBMED    25830891
REFERENCE   2  (bases 1 to 7090)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7090)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2015 Apr 1. doi: 10.1038/nature14299."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7090
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   42..182
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     enhancer        202..581
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        582..785
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     regulatory      804..813
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             816..836
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             861..4016
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="KRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4017..4064
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4071..4151
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_signal    4185..4392
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4405..4534
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(4596..5184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5358..6215)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6216..6320)
                     /label=AmpR promoter
     primer_bind     6388..6406
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     primer_bind     complement(6444..6466)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6566..6585
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     rep_origin      complement(6602..7057)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 accgcacaga tgcgtaagga gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc
       61 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc
      121 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
      181 ctgcggccgc ctcgaggcgt tgacattgat tattgactag ttattaatag taatcaatta
      241 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      301 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      361 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      421 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      481 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      541 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      601 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      661 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      721 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      781 gagctctctg gctaactacc ggtgccacca tggccccaaa gaagaagcgg aaggtcggta
      841 tccacggagt cccagcagcc aagcggaact acatcctggg cctggccatc ggcatcacca
      901 gcgtgggcta cggcatcatc gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc
      961 tgttcaaaga ggccaacgtg gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa
     1021 ggctgaagcg gcggaggcgg catagaatcc agagagtgaa gaagctgctg ttcgactaca
     1081 acctgctgac cgaccacagc gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg
     1141 gcctgagcca gaagctgagc gaggaagagt tctctgccgc cctgctgcac ctggccaaga
     1201 gaagaggcgt gcacaacgtg aacgaggtgg aagaggacac cggcaacgag ctgtccacca
     1261 aagagcagat cagccggaac agcaaggccc tggaagagaa atacgtggcc gaactgcagc
     1321 tggaacggct gaagaaagac ggcgaagtgc ggggcagcat caacagattc aagaccagcg
     1381 actacgtgaa agaagccaaa cagctgctga aggtgcagaa ggcctaccac cagctggacc
     1441 agagcttcat cgacacctac atcgacctgc tggaaacccg gcggacctac tatgagggac
     1501 ctggcgaggg cagccccttc ggctggaagg acatcaaaga atggtacgag atgctgatgg
     1561 gccactgcac ctacttcccc gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc
     1621 tgtacaacgc cctgaacgac ctgaacaatc tcgtgatcac cagggacgag aacgagaagc
     1681 tggaatatta cgagaagttc cagatcatcg agaacgtgtt caagcagaag aagaagccca
     1741 ccctgaagca gatcgccaaa gaaatcctcg tgaacgaaga ggatattaag ggctacagag
     1801 tgaccagcac cggcaagccc gagttcacca acctgaaggt gtaccacgac atcaaggaca
     1861 ttaccgcccg gaaagagatt attgagaacg ccgagctgct ggatcagatt gccaagatcc
     1921 tgaccatcta ccagagcagc gaggacatcc aggaagaact gaccaatctg aactccgagc
     1981 tgacccagga agagatcgag cagatctcta atctgaaggg ctataccggc acccacaacc
     2041 tgagcctgaa ggccatcaac ctgatcctgg acgagctgtg gcacaccaac gacaaccaga
     2101 tcgctatctt caaccggctg aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag
     2161 agatccccac caccctggtg gacgacttca tcctgagccc cgtcgtgaag agaagcttca
     2221 tccagagcat caaagtgatc aacgccatca tcaagaagta cggcctgccc aacgacatca
     2281 ttatcgagct ggcccgcgag aagaactcca aggacgccca gaaaatgatc aacgagatgc
     2341 agaagcggaa ccggcagacc aacgagcgga tcgaggaaat catccggacc accggcaaag
     2401 agaacgccaa gtacctgatc gagaagatca agctgcacga catgcaggaa ggcaagtgcc
     2461 tgtacagcct ggaagccatc cctctggaag atctgctgaa caaccccttc aactatgagg
     2521 tggaccacat catccccaga agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg
     2581 tgaagcagga agaagccagc aagaagggca accggacccc attccagtac ctgagcagca
     2641 gcgacagcaa gatcagctac gaaaccttca agaagcacat cctgaatctg gccaagggca
     2701 agggcagaat cagcaagacc aagaaagagt atctgctgga agaacgggac atcaacaggt
     2761 tctccgtgca gaaagacttc atcaaccgga acctggtgga taccagatac gccaccagag
     2821 gcctgatgaa cctgctgcgg agctacttca gagtgaacaa cctggacgtg aaagtgaagt
     2881 ccatcaatgg cggcttcacc agctttctgc ggcggaagtg gaagtttaag aaagagcgga
     2941 acaaggggta caagcaccac gccgaggacg ccctgatcat tgccaacgcc gatttcatct
     3001 tcaaagagtg gaagaaactg gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg
     3061 aaaagcaggc cgagagcatg cccgagatcg aaaccgagca ggagtacaaa gagatcttca
     3121 tcacccccca ccagatcaag cacattaagg acttcaagga ctacaagtac agccaccggg
     3181 tggacaagaa gcctaataga gagctgatta acgacaccct gtactccacc cggaaggacg
     3241 acaagggcaa caccctgatc gtgaacaatc tgaacggcct gtacgacaag gacaatgaca
     3301 agctgaaaaa gctgatcaac aagagccccg aaaagctgct gatgtaccac cacgaccccc
     3361 agacctacca gaaactgaag ctgattatgg aacagtacgg cgacgagaag aatcccctgt
     3421 acaagtacta cgaggaaacc gggaactacc tgaccaagta ctccaaaaag gacaacggcc
     3481 ccgtgatcaa gaagattaag tattacggca acaaactgaa cgcccatctg gacatcaccg
     3541 acgactaccc caacagcaga aacaaggtcg tgaagctgtc cctgaagccc tacagattcg
     3601 acgtgtacct ggacaatggc gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca
     3661 aaaaagaaaa ctactacgaa gtgaatagca agtgctatga ggaagctaag aagctgaaga
     3721 agatcagcaa ccaggccgag tttatcgcct ccttctacaa caacgatctg atcaagatca
     3781 acggcgagct gtatagagtg atcggcgtga acaacgacct gctgaaccgg atcgaagtga
     3841 acatgatcga catcacctac cgcgagtacc tggaaaacat gaacgacaag aggcccccca
     3901 ggatcattaa gacaatcgcc tccaagaccc agagcattaa gaagtacagc acagacattc
     3961 tgggcaacct gtatgaagtg aaatctaaga agcaccctca gatcatcaaa aagggcaaaa
     4021 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagggatcc tacccatacg
     4081 atgttccaga ttacgcttat ccctacgacg tgcctgatta tgcataccca tatgatgtcc
     4141 ccgactatgc ctaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     4201 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4261 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4321 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag
     4381 gcatgctggg gagctagagg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     4441 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggcggcc
     4501 tcagtgagcg agcgagcgcg cagctgcctg caggacatgt gagcaaaagg ccagcaaaag
     4561 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4621 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4681 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4741 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4801 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4861 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4921 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4981 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5041 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5101 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5161 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5221 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5281 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5341 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5401 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5461 ttaccatctg gccccagtgc tgcaatgata ccgcggcttc cacgctcacc ggctccagat
     5521 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5581 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5641 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5701 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5761 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5821 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5881 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5941 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6001 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6061 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6121 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6181 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6241 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6301 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6361 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     6421 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     6481 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     6541 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     6601 aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     6661 ttttttaacc aatagaccga aatcggcaaa atcccttata aatcaaaaga atagcccgag
     6721 atagagttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     6781 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     6841 aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     6901 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     6961 gcgaaaggag cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     7021 acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgtatgc
     7081 ggtgtgaaat
//
