LOCUS       40924_47063        7446 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 7446)
  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 7446)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7446)
  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..7446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             813..3968
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3969..4016
                     /codon_start=1
                     /product="bipartite nuclear localization signal from
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4023..4103
                     /codon_start=1
                     /label=3xHA
                     /note="three tandem HA epitope tags"
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     polyA_signal    4137..4344
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4351..4591
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4620..4695
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4709..4849
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      4924..5379
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5396..5415)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     5515..5537
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5575..5593)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        5661..5765
                     /label=AmpR promoter
     CDS             5766..6623
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6797..7385
                     /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 gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca
      841 tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg
      901 ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga
      961 gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc
     1021 tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg
     1081 ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc
     1141 acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg
     1201 agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg
     1261 ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat
     1321 tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc
     1381 accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct
     1441 actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg
     1501 agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct
     1561 acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg
     1621 agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga
     1681 agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta
     1741 agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg
     1801 acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga
     1861 ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc
     1921 tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg
     1981 gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca
     2041 acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt
     2101 cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga
     2161 agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc
     2221 ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga
     2281 tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga
     2341 ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg
     2401 aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct
     2461 tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca
     2521 acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt
     2581 acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc
     2641 tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg
     2701 acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat
     2761 acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg
     2821 tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta
     2881 agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg
     2941 ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc
     3001 agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca
     3061 aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt
     3121 acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca
     3181 cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca
     3241 aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc
     3301 accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga
     3361 agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa
     3421 aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc
     3481 tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc
     3541 cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc
     3601 tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta
     3661 agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc
     3721 tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc
     3781 ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca
     3841 agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca
     3901 gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca
     3961 aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggat
     4021 cctacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttacc
     4081 catacgatgt tccagattac gcttaagaat tcctagagct cgctgatcag cctcgactgt
     4141 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     4201 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     4261 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     4321 agagaatagc aggcatgctg gggaggtacc gagggcctat ttcccatgat tccttcatat
     4381 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
     4441 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
     4501 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
     4561 ttcttggctt tatatatctt gtggaaagga cgaaacaccg gagaccacgg caggtctcag
     4621 ttttagtact ctggaaacag aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc
     4681 aacttgttgg cgagattttt gcggccgcag gaacccctag tgatggagtt ggccactccc
     4741 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     4801 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     4861 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     4921 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4981 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5041 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5101 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     5161 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5221 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     5281 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     5341 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     5401 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     5461 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     5521 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     5581 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     5641 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5701 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5761 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5821 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5881 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5941 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     6001 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     6061 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     6121 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     6181 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     6241 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     6301 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     6361 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     6421 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     6481 gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     6541 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     6601 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6661 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6721 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6781 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6841 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6901 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6961 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     7021 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     7081 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     7141 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     7201 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     7261 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     7321 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     7381 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     7441 acatgt
//
