LOCUS       40924_47083        7293 bp DNA     circular SYN 13-MAY-2021
DEFINITION  A single vector AAV-Cas9 system containing Cas9 from Staphylococcus 
            aureus (SaCas9) and its sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7293)
  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 7293)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7293)
  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..7293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        187..596
                     /label=TBG promoter
                     /note="promoter from the human thyroxine-binding globulin
                     gene (Yan et al., 2012)"
     regulatory      615..624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             627..647
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             672..3827
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTREQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3828..3875
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3882..3908
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             3909..3950
                     /codon_start=1
                     /label=OLLAS tag
                     /note="Escherichia coli OmpF Linker and mouse Langerin
                     fusion sequence tag (Park et al., 2008)"
                     /translation="SGFANELGPRLMGK"
     polyA_signal    3984..4191
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4203..4278)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(4307..4547)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     repeat_region   4556..4696
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4771..5226
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5243..5262)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     5362..5384
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5422..5440)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        5508..5612
                     /label=AmpR promoter
     CDS             5613..6470
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6644..7232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggg gctggaagct acctttgaca tcatttcctc
      181 tgcgaatgca tgtataattt ctacagaacc tattagaaag gatcacccag cctctgcttt
      241 tgtacaactt tcccttaaaa aactgccaat tccactgctg tttggcccaa tagtgagaac
      301 tttttcctgc tgcctcttgg tgcttttgcc tatggcccct attctgcctg ctgaagacac
      361 tcttgccagc atggacttaa acccctccag ctctgacaat cctctttctc ttttgtttta
      421 catgaagggt ctggcagcca aagcaatcac tcaaagttca aaccttatca ttttttgctt
      481 tgttcctctt ggccttggtt ttgtacatca gctttgaaaa taccatccca gggttaatgc
      541 tggggttaat ttataactaa gagtgctcta gttttgcaat acaggacatg ctataaaaat
      601 ggaaagatac cggtgccacc atggccccaa agaagaagcg gaaggtcggt atccacggag
      661 tcccagcagc caagcggaac tacatcctgg gcctggacat cggcatcacc agcgtgggct
      721 acggcatcat cgactacgag acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag
      781 aggccaacgt ggaaaacaac gagggcaggc ggagcaagag aggcgccaga aggctgaagc
      841 ggcggaggcg gcatagaatc cagagagtga agaagctgct gttcgactac aacctgctga
      901 ccgaccacag cgagctgagc ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc
      961 agaagctgag cgaggaagag ttctctgccg ccctgctgca cctggccaag agaagaggcg
     1021 tgcacaacgt gaacgaggtg gaagaggaca ccggcaacga gctgtccacc agagagcaga
     1081 tcagccggaa cagcaaggcc ctggaagaga aatacgtggc cgaactgcag ctggaacggc
     1141 tgaagaaaga cggcgaagtg cggggcagca tcaacagatt caagaccagc gactacgtga
     1201 aagaagccaa acagctgctg aaggtgcaga aggcctacca ccagctggac cagagcttca
     1261 tcgacaccta catcgacctg ctggaaaccc ggcggaccta ctatgaggga cctggcgagg
     1321 gcagcccctt cggctggaag gacatcaaag aatggtacga gatgctgatg ggccactgca
     1381 cctacttccc cgaggaactg cggagcgtga agtacgccta caacgccgac ctgtacaacg
     1441 ccctgaacga cctgaacaat ctcgtgatca ccagggacga gaacgagaag ctggaatatt
     1501 acgagaagtt ccagatcatc gagaacgtgt tcaagcagaa gaagaagccc accctgaagc
     1561 agatcgccaa agaaatcctc gtgaacgaag aggatattaa gggctacaga gtgaccagca
     1621 ccggcaagcc cgagttcacc aacctgaagg tgtaccacga catcaaggac attaccgccc
     1681 ggaaagagat tattgagaac gccgagctgc tggatcagat tgccaagatc ctgaccatct
     1741 accagagcag cgaggacatc caggaagaac tgaccaatct gaactccgag ctgacccagg
     1801 aagagatcga gcagatctct aatctgaagg gctataccgg cacccacaac ctgagcctga
     1861 aggccatcaa cctgatcctg gacgagctgt ggcacaccaa cgacaaccag atcgctatct
     1921 tcaaccggct gaagctggtg cccaagaagg tggacctgtc ccagcagaaa gagatcccca
     1981 ccaccctggt ggacgacttc atcctgagcc ccgtcgtgaa gagaagcttc atccagagca
     2041 tcaaagtgat caacgccatc atcaagaagt acggcctgcc caacgacatc attatcgagc
     2101 tggcccgcga gaagaactcc aaggacgccc agaaaatgat caacgagatg cagaagcgga
     2161 accggcagac caacgagcgg atcgaggaaa tcatccggac caccggcaaa gagaacgcca
     2221 agtacctgat cgagaagatc aagctgcacg acatgcagga aggcaagtgc ctgtacagcc
     2281 tggaagccat ccctctggaa gatctgctga acaacccctt caactatgag gtggaccaca
     2341 tcatccccag aagcgtgtcc ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg
     2401 aagaaaacag caagaagggc aaccggaccc cattccagta cctgagcagc agcgacagca
     2461 agatcagcta cgaaaccttc aagaagcaca tcctgaatct ggccaagggc aagggcagaa
     2521 tcagcaagac caagaaagag tatctgctgg aagaacggga catcaacagg ttctccgtgc
     2581 agaaagactt catcaaccgg aacctggtgg ataccagata cgccaccaga ggcctgatga
     2641 acctgctgcg gagctacttc agagtgaaca acctggacgt gaaagtgaag tccatcaatg
     2701 gcggcttcac cagctttctg cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt
     2761 acaagcacca cgccgaggac gccctgatca ttgccaacgc cgatttcatc ttcaaagagt
     2821 ggaagaaact ggacaaggcc aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg
     2881 ccgagagcat gcccgagatc gaaaccgagc aggagtacaa agagatcttc atcacccccc
     2941 accagatcaa gcacattaag gacttcaagg actacaagta cagccaccgg gtggacaaga
     3001 agcctaatag agagctgatt aacgacaccc tgtactccac ccggaaggac gacaagggca
     3061 acaccctgat cgtgaacaat ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa
     3121 agctgatcaa caagagcccc gaaaagctgc tgatgtacca ccacgacccc cagacctacc
     3181 agaaactgaa gctgattatg gaacagtacg gcgacgagaa gaatcccctg tacaagtact
     3241 acgaggaaac cgggaactac ctgaccaagt actccaaaaa ggacaacggc cccgtgatca
     3301 agaagattaa gtattacggc aacaaactga acgcccatct ggacatcacc gacgactacc
     3361 ccaacagcag aaacaaggtc gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc
     3421 tggacaatgg cgtgtacaag ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa
     3481 actactacga agtgaatagc aagtgctatg aggaagctaa gaagctgaag aagatcagca
     3541 accaggccga gtttatcgcc tccttctaca acaacgatct gatcaagatc aacggcgagc
     3601 tgtatagagt gatcggcgtg aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg
     3661 acatcaccta ccgcgagtac ctggaaaaca tgaacgacaa gaggcccccc aggatcatta
     3721 agacaatcgc ctccaagacc cagagcatta agaagtacag cacagacatt ctgggcaacc
     3781 tgtatgaagt gaaatctaag aagcaccctc agatcatcaa aaagggcaaa aggccggcgg
     3841 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ctacccatac gatgttccag
     3901 attacgctag cggcttcgcc aacgagcttg gacccaggtt gatgggaaag taagaattcc
     3961 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4021 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4081 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4141 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg aggtaccaaa
     4201 aatctcgcca acaagttgac gagataaaca cggcattttg ccttgtttta gtagattctg
     4261 tttccagagt actaaaactg agacctgccg tggtctccgg tgtttcgtcc tttccacaag
     4321 atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt
     4381 ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta
     4441 ttttgtacta atatctttgt gtttacagtc aaattaattc caattatctc tctaacagcc
     4501 ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcgcg gccgcaggaa
     4561 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg
     4621 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg
     4681 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4741 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
     4801 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     4861 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     4921 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     4981 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     5041 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     5101 accctatctc gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     5161 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     5221 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     5281 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5341 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5401 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     5461 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5521 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5581 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5641 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5701 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5761 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5821 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5881 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5941 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6001 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6061 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6121 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6181 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6241 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6301 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     6361 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6421 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6481 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6541 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6601 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6661 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6721 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6781 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6841 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6901 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6961 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7021 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7081 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7141 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7201 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7261 tcctggcctt ttgctggcct tttgctcaca tgt
//
