LOCUS       sequence-421040-        7446 bp    DNA     circular SYN 14-APR-2025
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)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2015 Apr 1. doi: 10.1038/nature14299."
COMMENT     Sequence Label: 
            pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA
FEATURES             Location/Qualifiers
     source          1..7446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(869..889)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      892..901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(920..1123)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(949..969)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1124..1503
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   1527..1656
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1718..2306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1798..1817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2477..3268,3269..3337))
                     /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     3100..3119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3338..3442)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3510..3528
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3566..3588)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3688..3707
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3724..4179)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3861..3882)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4073..4092
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4254..4394
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4265..4394
                     /label=AAV2 ITR
     misc_RNA        complement(4408..4483)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(4504..4752)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4562..4581)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4732..4752)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    4759..4966
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4955..4972
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5000..5080)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(5087..5134)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(5135..7446,1..844))
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
ORIGIN
        1 tctggaactt ctcgtaatat tccagcttct cgttctcgtc cctggtgatc acgagattgt
       61 tcaggtcgtt cagggcgttg tacaggtcgg cgttgtaggc gtacttcacg ctccgcagtt
      121 cctcggggaa gtaggtgcag tggcccatca gcatctcgta ccattctttg atgtccttcc
      181 agccgaaggg gctgccctcg ccaggtccct catagtaggt ccgccgggtt tccagcaggt
      241 cgatgtaggt gtcgatgaag ctctggtcca gctggtggta ggccttctgc accttcagca
      301 gctgtttggc ttctttcacg tagtcgctgg tcttgaatct gttgatgctg ccccgcactt
      361 cgccgtcttt cttcagccgt tccagctgca gttcggccac gtatttctct tccagggcct
      421 tgctgttccg gctgatctgc tctttggtgg acagctcgtt gccggtgtcc tcttccacct
      481 cgttcacgtt gtgcacgcct cttctcttgg ccaggtgcag cagggcggca gagaactctt
      541 cctcgctcag cttctggctc aggcccttca ctctggcctc gtaggggttg atgccgctca
      601 gctcgctgtg gtcggtcagc aggttgtagt cgaacagcag cttcttcact ctctggattc
      661 tatgccgcct ccgccgcttc agccttctgg cgcctctctt gctccgcctg ccctcgttgt
      721 tttccacgtt ggcctctttg aacagccgca cgccggcatc gatcacgtcc cgtgtctcgt
      781 agtcgatgat gccgtagccc acgctggtga tgccgatgtc caggcccagg atgtagttcc
      841 gcttggctgc tgggactccg tggataccga ccttccgctt cttctttggg gccatggtgg
      901 caccggtagt tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc
      961 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
     1021 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
     1081 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
     1141 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     1201 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     1261 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     1321 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     1381 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     1441 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     1501 gtcaacgcct cgagtctaga ggccgcagga acccctagtg atggagttgg ccactccctc
     1561 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggcgg
     1621 cctcagtgag cgagcgagcg cgcagctgcc tgcaggacat gtgagcaaaa ggccagcaaa
     1681 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1741 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1801 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1861 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1921 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1981 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2041 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2101 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2161 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2221 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2281 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2341 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2401 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2461 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2521 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2581 gcttaccatc tggccccagt gctgcaatga taccgcggct tccacgctca ccggctccag
     2641 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2701 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2761 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     2821 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2881 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2941 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3001 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3061 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3121 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3181 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3241 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3301 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3361 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3421 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     3481 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     3541 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     3601 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     3661 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     3721 ataaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3781 cattttttaa ccaatagacc gaaatcggca aaatccctta taaatcaaaa gaatagcccg
     3841 agatagagtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3901 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3961 ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     4021 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     4081 aagcgaaagg agcgggcgct aaggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     4141 ccacacccgc cgcgcttaat gcgccgctac agggcgcgta ctatggttgc tttgacgtat
     4201 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gcccctgcag
     4261 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac
     4321 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     4381 cactaggggt tcctgcggcc gcaaaaatct cgccaacaag ttgacgagat aaacacggca
     4441 ttttgccttg ttttagtaga ttctgtttcc agagtactaa aactgagacc tgccgtggtc
     4501 tccggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt
     4561 tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag
     4621 aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt
     4681 aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg
     4741 aaataggccc tcggtacctc cccagcatgc ctgctattct cttcccaatc ctcccccttg
     4801 ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca
     4861 atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc
     4921 acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag
     4981 cgagctctag gaattcttaa gcgtaatctg gaacatcgta tgggtaagcg taatctggaa
     5041 catcgtatgg gtaagcgtaa tctggaacat cgtatgggta ggatcccttt ttcttttttg
     5101 cctggccggc ctttttcgtg gccgccggcc ttttgccctt tttgatgatc tgagggtgct
     5161 tcttagattt cacttcatac aggttgccca gaatgtctgt gctgtacttc ttaatgctct
     5221 gggtcttgga ggcgattgtc ttaatgatcc tggggggcct cttgtcgttc atgttttcca
     5281 ggtactcgcg gtaggtgatg tcgatcatgt tcacttcgat ccggttcagc aggtcgttgt
     5341 tcacgccgat cactctatac agctcgccgt tgatcttgat cagatcgttg ttgtagaagg
     5401 aggcgataaa ctcggcctgg ttgctgatct tcttcagctt cttagcttcc tcatagcact
     5461 tgctattcac ttcgtagtag ttttcttttt tgatcacatc cagattcttc acggtcacga
     5521 acttgtacac gccattgtcc aggtacacgt cgaatctgta gggcttcagg gacagcttca
     5581 cgaccttgtt tctgctgttg gggtagtcgt cggtgatgtc cagatgggcg ttcagtttgt
     5641 tgccgtaata cttaatcttc ttgatcacgg ggccgttgtc ctttttggag tacttggtca
     5701 ggtagttccc ggtttcctcg tagtacttgt acaggggatt cttctcgtcg ccgtactgtt
     5761 ccataatcag cttcagtttc tggtaggtct gggggtcgtg gtggtacatc agcagctttt
     5821 cggggctctt gttgatcagc tttttcagct tgtcattgtc cttgtcgtac aggccgttca
     5881 gattgttcac gatcagggtg ttgcccttgt cgtccttccg ggtggagtac agggtgtcgt
     5941 taatcagctc tctattaggc ttcttgtcca cccggtggct gtacttgtag tccttgaagt
     6001 ccttaatgtg cttgatctgg tggggggtga tgaagatctc tttgtactcc tgctcggttt
     6061 cgatctcggg catgctctcg gcctgctttt cctcgaacat ctggttttcc atcacttttt
     6121 tggccttgtc cagtttcttc cactctttga agatgaaatc ggcgttggca atgatcaggg
     6181 cgtcctcggc gtggtgcttg taccccttgt tccgctcttt cttaaacttc cacttccgcc
     6241 gcagaaagct ggtgaagccg ccattgatgg acttcacttt cacgtccagg ttgttcactc
     6301 tgaagtagct ccgcagcagg ttcatcaggc ctctggtggc gtatctggta tccaccaggt
     6361 tccggttgat gaagtctttc tgcacggaga acctgttgat gtcccgttct tccagcagat
     6421 actctttctt ggtcttgctg attctgccct tgcccttggc cagattcagg atgtgcttct
     6481 tgaaggtttc gtagctgatc ttgctgtcgc tgctgctcag gtactggaat ggggtccggt
     6541 tgcccttctt gctgttttct tcctgcttca cgagcacctt gttgttgaag ctgttgtcga
     6601 aggacacgct tctggggatg atgtggtcca cctcatagtt gaaggggttg ttcagcagat
     6661 cttccagagg gatggcttcc aggctgtaca ggcacttgcc ttcctgcatg tcgtgcagct
     6721 tgatcttctc gatcaggtac ttggcgttct ctttgccggt ggtccggatg atttcctcga
     6781 tccgctcgtt ggtctgccgg ttccgcttct gcatctcgtt gatcattttc tgggcgtcct
     6841 tggagttctt ctcgcgggcc agctcgataa tgatgtcgtt gggcaggccg tacttcttga
     6901 tgatggcgtt gatcactttg atgctctgga tgaagcttct cttcacgacg gggctcagga
     6961 tgaagtcgtc caccagggtg gtggggatct ctttctgctg ggacaggtcc accttcttgg
     7021 gcaccagctt cagccggttg aagatagcga tctggttgtc gttggtgtgc cacagctcgt
     7081 ccaggatcag gttgatggcc ttcaggctca ggttgtgggt gccggtatag cccttcagat
     7141 tagagatctg ctcgatctct tcctgggtca gctcggagtt cagattggtc agttcttcct
     7201 ggatgtcctc gctgctctgg tagatggtca ggatcttggc aatctgatcc agcagctcgg
     7261 cgttctcaat aatctctttc cgggcggtaa tgtccttgat gtcgtggtac accttcaggt
     7321 tggtgaactc gggcttgccg gtgctggtca ctctgtagcc cttaatatcc tcttcgttca
     7381 cgaggatttc tttggcgatc tgcttcaggg tgggcttctt cttctgcttg aacacgttct
     7441 cgatga
//
