LOCUS       Exported                7464 bp ds-DNA     circular SYN 16-SEP-2022
DEFINITION  A single vector AAV-Cas9 system containing Cas9 from Staphylococcus 
            aureus (SaCas9) and its sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7464)
  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 7464)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Sep 16, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7464
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      756..765
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     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..3986
                     /codon_start=1
                     /label=SpeCas9
                     /translation="MATNYILGLDIGITSVGYGIINYEDKTIIDAGVRLFPEANVENNE
                     GRRSKRGARRLKRRRIHRLDRVKQLLNEYKLVKLNDIPKSTNPYEIRVKGLREELTRDE
                     LVIALLHLAKRRGIHNIEVIEQDNEEGNQLSTKEQLSKNNNLLKDKFVCELLLERFNDG
                     KVRGEENRFKTSDIIKEAKQILEVQKDVHNLDDYFINKYIELVETRREYYEGPGEGSPF
                     GWDGDLKKWYEMLMGRCTYFPEELASVKYAYSADLFNALNDLNNLVIQRDGSTKLEYHE
                     KYHIIENVFKQKKKPTLKQIANEIEVTPEDIKGYRITKSGKENFTEFKLYHDLKKVLKD
                     QSILENVSLLDQIAEILTVYQDKESIKKELSKLDETINDIDKESISNLTGYNGTHRLSL
                     KCINLVLEELWHSSRAQMEIFAYLNIKPKKIDLKKSNKIPKDMIDEFILSPVVKRTFGQ
                     AINVINKVIEKYGVPNDIIIELARENNSKDKQKFINELQKKNEKTRQRINEIIGEYGNQ
                     NAKRLVEKIRLHDEQEGKCLYSLESIPLDDLLNNPNYYEVDHIIPRSVSFDNSYQNKVL
                     VKQTENSKKGNRTPYQYLNSGEAKISYNQFKQHILNLSKSKDRISKKKKEYLLEERDIN
                     KFEVKKEFINRNLVDTRYATRELSNYLKAYFSANDMNVKIKTINGSFTDYLRKVWKFKK
                     ERNHGYKHHAEDALIIANADFLFKENKKLKEVNKVLEKPADNNELNKVENSIGIKTEDE
                     YKELFNIPQQVADIKEFKDFKFSHRVDKKPNRQLINDTLYSTRQLGEDSYIVQTIKNIY
                     SKDNTDLKKHFNKNPEKFLMYQHDPKTFEKLETIMKQYSNEKNPLAKYHEETGEYLTKY
                     SKKNNGPIVKTIKMLGNKVGNHLDVTHKYKNSNKQLVRLSIKSLRFDVYLTDKGYKFIT
                     ISYLDILKKDSYYYIPIETYEKLKTNKGINKSSQFIGSFYYNDLIELDGEVYKIIGVNS
                     DKRNIIELDLPNIRYKEYCELNNVKGEARIKKSIGKKVQSIKKLSTDVLGNRYYQKNVQ
                     NPQLLFKRGN"
     primer_bind     complement(1533..1565)
                     /label=Sa-A-R246A-R
     primer_bind     1551..1571
                     /label=Sa-A-R246A-F
     primer_bind     complement(2037..2075)
                     /label=Sa-A-N414A-S420A-R
     primer_bind     complement(2037..2072)
                     /label=Sa-A-N414A-R2
     primer_bind     2055..2093
                     /label=Sa-A-N414A-S420A-F
     primer_bind     complement(2760..2792)
                     /label=Sa-A-R655A-R
     primer_bind     2781..2798
                     /label=Sa-A-R655A-F
     primer_bind     3099..3118
                     /label=Sa-A-qPCR-F
     primer_bind     complement(3210..3229)
                     /label=Sa-A-qPCR-R
     primer_bind     complement(3757..3776)
                     /label=SpeCas9-S989N-R
     primer_bind     3779..3797
                     /label=SpeCas9-S989N-F
     CDS             3987..4034
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4041..4121
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4149..4166)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4155..4362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4369..4609
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4369..4389
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4540..4559
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4638..4713
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4727..4867
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4727..4856
                     /label=AAV2 ITR
     rep_origin      4942..5397
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5029..5048)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5239..5260
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5414..5433)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5533..5555
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5593..5611)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5679..5783
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5784..6644
                     /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     complement(6002..6021)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6815..7403
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7304..7323
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
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 ccatggccac aaactacatc ttgggcctgg
      841 atatcggaat cacaagcgtg ggatacggca tcatcaacta tgaggacaag accatcatcg
      901 acgccggcgt gcggctgttt ccagaggcca atgtggaaaa taacgaaggc agaagaagca
      961 agagaggcgc cagacgttta aagcggcggc ggatccacag actggaccgg gtgaagcagc
     1021 tgctgaacga gtacaagctg gtgaagctga atgacatccc caagtccacc aacccttatg
     1081 agatccgcgt gaagggcctg agagaggaac tgacacggga tgagctggtg atcgccctgc
     1141 tgcacctggc caagcggaga ggaatccaca atatcgaggt tatcgagcag gacaacgagg
     1201 aagggaacca gctgagcaca aaggagcagt tgtctaagaa caacaacctg ctgaaggata
     1261 agttcgtgtg tgaactgctg ctggaaagat ttaacgacgg caaggtgcgg ggcgaggaaa
     1321 ataggttcaa gacctccgac atcattaagg aagctaagca gattctggaa gtgcaaaagg
     1381 acgtgcacaa cctggacgat tacttcatca acaagtacat cgagctggtc gagacaagac
     1441 gggaatatta cgagggcccc ggagaaggct ctcctttcgg ctgggacggc gacctgaaga
     1501 aatggtacga gatgctgatg ggcagatgta cctactttcc tgaggagctg gcctctgtga
     1561 aatacgctta cagcgccgac ctgttcaacg ccctgaacga cctgaacaat ctggtgatcc
     1621 agcgggacgg ctctacgaaa ctggaatacc acgagaagta ccacatcatc gaaaacgtgt
     1681 tcaagcagaa gaagaagcct acactgaaac agatcgctaa cgagatcgag gtgacccctg
     1741 aagatatcaa gggctataga atcacaaaga gcggcaagga aaatttcaca gagttcaaac
     1801 tgtatcacga tctgaaaaag gtgctgaagg accagagcat cctggaaaac gtttctctgc
     1861 tggatcagat cgccgaaatc ctgaccgtgt accaggataa agaatccatc aagaaagagc
     1921 tgtccaagct ggacgaaacc atcaatgata ttgacaagga aagcatcagc aacctgacag
     1981 gctataacgg cacccaccgg ctgtctctga agtgcatcaa cctggtccta gaagagctgt
     2041 ggcacagcag cagagcccaa atggaaattt tcgcctacct gaacatcaag cctaaaaaga
     2101 tcgatctgaa gaagagcaac aagatcccta aggacatgat cgacgagttc atcctgagcc
     2161 ctgtggtgaa gagaacattt ggccaggcca tcaacgtgat caacaaggtg atcgagaaat
     2221 acggagtgcc caacgacatc atcatcgaac tggccagaga gaataacagc aaggacaagc
     2281 agaaattcat caacgagctt cagaagaaga acgagaagac ccggcagaga atcaatgaga
     2341 tcatcggaga atacggcaat cagaacgcta agcggctggt ggaaaagatc agactgcacg
     2401 acgaacagga gggaaagtgc ctgtactctc tggaaagcat cccactggat gatcttctga
     2461 ataatccaaa ttactacgag gtggaccaca tcatccctag aagcgtgtct ttcgacaaca
     2521 gctaccaaaa caaagtgctg gtgaagcaga ccgagaatag caagaaaggc aacagaaccc
     2581 cttaccagta cctgaactcc ggcgaggcca agatcagcta caatcagttt aagcagcaca
     2641 tcctgaacct gtcaaagtct aaggacagaa tctcaaagaa gaagaaggaa tacctgctgg
     2701 aagagcggga cattaacaag ttcgaggtga agaaggaatt catcaacaga aacctcgttg
     2761 ataccagata cgccaccaga gagctgagca actacctgaa ggcctacttc tccgccaacg
     2821 acatgaacgt aaagatcaag acaatcaacg gcagcttcac cgactacctc agaaaggtgt
     2881 ggaagttcaa gaaggaacgg aaccacggct acaagcacca cgccgaggac gcactgatca
     2941 tcgctaatgc cgacttcctg ttcaaggaaa acaaaaaact caaggaagtg aacaaagtgc
     3001 tagagaagcc cgccgacaat aacgaactta ataaagtcga gaactctatc ggcatcaaaa
     3061 ccgaggatga gtacaaggag ctgtttaaca tcccccagca ggtggccgac atcaaagagt
     3121 tcaaggactt caagtttagc caccgcgtgg ataagaaacc aaaccggcaa ctaatcaacg
     3181 acaccctgta cagcaccaga cagctgggcg aggacagcta catcgtgcag accatcaaaa
     3241 acatttacag caaggacaac accgatctga agaagcattt caacaaaaat cctgagaagt
     3301 tcctgatgta ccagcatgat cccaagacct tcgagaagct ggaaaccatc atgaaacaat
     3361 acagcaatga aaagaacccc ctggccaagt accatgagga aaccggcgag tacctgacca
     3421 agtatagtaa gaagaacaac ggccctatcg tgaagacaat caagatgctg ggcaacaagg
     3481 tgggcaatca cctggacgtg acccacaagt acaagaacag caataagcaa ctggtgaggc
     3541 tgtccataaa gtccctcaga tttgacgtgt accttacaga caagggatac aagttcatca
     3601 ccatctctta cctggacatc ctgaaaaaag acagctacta ctacatccct attgaaacct
     3661 acgagaagct gaaaacaaac aagggcatca acaagtcgag ccagttcatc ggcagcttct
     3721 actacaacga cctgatcgag ctggatggcg aagtgtacaa gatcataggt gtgaacagcg
     3781 acaagcgcaa catcattgag ctggacctgc ctaacatcag atacaaggaa tactgcgagc
     3841 tgaacaacgt gaagggagaa gccagaatca agaagtccat aggcaagaag gtgcagagca
     3901 tcaaaaagct gagcaccgac gtgctgggaa acagatacta tcagaagaac gtccagaacc
     3961 ctcagctgct gtttaaacgg ggcaacaaaa ggccggcggc cacgaaaaag gccggccagg
     4021 caaaaaagaa aaagggatcc tacccatacg atgttccaga ttacgcttac ccatacgatg
     4081 ttccagatta cgcttaccca tacgatgttc cagattacgc ttaagaattc ctagagctcg
     4141 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4201 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4261 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4321 caagggggag gattgggaag agaatagcag gcatgctggg gaggtaccga gggcctattt
     4381 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     4441 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     4501 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     4561 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgga
     4621 gaccacggca ggtctcagtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa
     4681 tgccgtgttt atctcgtcaa cttgttggcg agatttttgc ggccgcagga acccctagtg
     4741 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4801 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4861 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4921 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4981 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     5041 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     5101 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     5161 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     5221 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     5281 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     5341 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5401 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5461 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5521 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5581 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5641 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5701 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5761 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5821 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5881 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5941 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6001 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6061 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6121 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6181 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6241 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6301 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6361 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6421 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6481 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     6541 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6601 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6661 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6721 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6781 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6841 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6901 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6961 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7021 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7081 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7141 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7201 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7261 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7321 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7381 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7441 tttgctggcc ttttgctcac atgt
//
