LOCUS       Exported                7461 bp ds-DNA     circular SYN 29-MAR-2021
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 7461)
  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 7461)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Sep 16, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7461
                     /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..3983
                     /codon_start=1
                     /label=Sha2Cas9
                     /translation="TTNYILGLDIGITSVGYGIINYEDKTIIDAGVRLFPEANVENNEG
                     RRSKRGARRLKRRRIHRLDRIKQLLNEYKLVELNDVPKSTNPYDIRVKGLKEELTREEL
                     VIALLHLAKRRGIHNIDVVEQDGEEGNQLSTKEQLSKNKNLLKDKFVCELLLERFNEGK
                     VRGEENRFKTSDIIKEAEQILKVQKNIHNLDEHFINKYIELVKTRREYFEGPGEGSPFG
                     WNGDLKKWYEMLMGHCTYFPEELRSVKYAYSADLFNALNDLNNLVIQRDGSTKLEYYEK
                     YHIIENVFKQKKKPTLKQIANEIGVTPEDIKGYRITKSGKENFTEFKLYHDLKKVLKDQ
                     SILENVSLLDQIAEILTVYQDKGSIKNELSKLDETINETDKENISNLTGYNGTHRLSLK
                     CINLVLEELWHSSRNQMEIFSYLNIKPKKIDLKKSNKIPKDMIDEFILSPVVKRTFGQA
                     INVINKIIEKYGIPNDIIIELARENNSKDKQKFINELQRKNEKTRQRINEIIGEYGNQN
                     AKRLVEKIKLHDEQEGKCLYSLESIPLEDLLNNPNYYEVDHIIPRSVSFDNSYQNKVLV
                     KQTENSKKGNRTPYQYLNSGEAKISYNQFKQHILNLSKSKDRISKKKKEYLLEERDINK
                     FEVKKEFINRNLVDTRYATRELSNYLKAYFSANDMNVKIKTINGSFTDYLRKVWKFKKE
                     RNHGYKHHAEDALIIANADFLFKENKKLKEANKVLEKPTESNELNKVENSTRIKNEDEY
                     KELFNIPQQVTDIKEFKDFKFSHRVDKKPNRQLINDTLYSTRQVGDDAYIVQTIKNIYS
                     KDNTDLKKHFNKNPEKFLMYQHDPKTFEKLETIMKQYSNEKNPLAKYHEETGEYLTKYS
                     KKDNGPIVKTIKMLSNKVGNHLDVTHKYEDSKKQLVRLSIKSLRFDVYLTDKGYKFITI
                     SYLDILKKDSYYYIPTETYNNLKANKSIDKSSQFIGSFYYNDLIELDGEVYKIIGVNSD
                     KRNLIELDLPNIRYKEYCELNNVKGEARIKKTIGKKVKSIKILHTDVLGNIYYQKNIQK
                     PQLLFKRGN"
     primer_bind     complement(1528..1562)
                     /label=Sa-10-HF-R246A-R
     primer_bind     1551..1568
                     /label=Sa-10-HF-R246A-F
     primer_bind     complement(2034..2069)
                     /label=Sa-10-N414A-S420A-R
     primer_bind     2054..2090
                     /label=Sa-10-N414A-S420A-F
     primer_bind     complement(2757..2789)
                     /label=Sa-10-R655A-R
     primer_bind     2778..2795
                     /label=Sa-10-R655A-F
     primer_bind     complement(3177..3194)
                     /label=Sa-10-R655A-R
     primer_bind     3433..3452
                     /label=Sa-10-qPCR-F
     primer_bind     complement(3565..3584)
                     /label=Sa-10-qPCR-R
     CDS             3984..4031
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4038..4118
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4146..4163)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4152..4359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4366..4606
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     4366..4386
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4537..4556
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4635..4710
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4724..4864
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4724..4853
                     /label=AAV2 ITR
     rep_origin      4939..5394
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5026..5045)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5236..5257
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5411..5430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5530..5552
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5590..5608)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5676..5780
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5781..6641
                     /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(5999..6018)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6812..7400
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7301..7320
                     /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 ccaccaccaa ctacatcctg ggcctggata
      841 tcggcatcac cagcgtgggc tacggcatca ttaactacga agataagacc atcatcgacg
      901 ccggcgtgag actgttcccc gaggctaatg tggaaaacaa cgaaggtaga agaagcaagc
      961 ggggcgcccg gagactgaag cggagaagaa tccacagact ggatagaatc aaacagctgc
     1021 tcaatgaata caagctggtt gagctgaacg acgtgccgaa gagcacaaac ccctacgata
     1081 tcagagtgaa gggcctgaaa gaggaactga caagagaaga gctcgtgatc gctctgctgc
     1141 acctggctaa gagaagaggc atccacaaca tcgacgtggt ggagcaggac ggcgaagagg
     1201 gaaaccagct gagcaccaag gagcagctgt ctaagaataa gaacctgctg aaggataagt
     1261 tcgtgtgcga gcttctgctg gaaagattca acgagggcaa ggtgcggggc gaagagaatc
     1321 gcttcaagac gagcgacatc atcaaggaag ccgagcaaat cctgaaggtt cagaagaata
     1381 tccataatct ggacgagcac ttcatcaata aatacattga gctggtgaag acccgcagag
     1441 aatattttga aggccctggc gagggcagcc ctttcggctg gaacggcgac ctgaaaaagt
     1501 ggtacgagat gctgatgggc cactgcacct acttccctga ggaactgaga tccgtgaaat
     1561 acgcctactc ggccgatctc tttaacgccc tgaacgactt aaacaacctg gtgatccaaa
     1621 gagatggatc aacaaagctg gaatactatg aaaagtatca tatcatcgag aacgtgttca
     1681 agcaaaagaa aaagcccacc ctgaaacaga tcgctaacga gatcggcgtc acccctgagg
     1741 acatcaaggg ctacagaatc accaagtccg gcaaggaaaa cttcaccgaa ttcaaactgt
     1801 accacgacct gaaaaaagtc ctgaaggacc agagcatcct ggagaacgtg agcctgctgg
     1861 accagatcgc cgagatcctc acagtgtacc aggacaaagg ctcgatcaag aatgagctga
     1921 gcaagctgga cgagacaatt aacgaaaccg acaaggaaaa catcagcaac ctgaccggct
     1981 acaacggcac acaccggctg agcctcaagt gtatcaacct ggtgttggag gaactgtggc
     2041 acagcagcag aaaccaaatg gagatcttct cttatctcaa catcaaacct aagaagatcg
     2101 acctcaaaaa gagcaacaaa atccctaagg acatgatcga cgagttcatt ctgtctcctg
     2161 tggtgaagcg gacattcggc caggccatca atgtgatcaa caaaatcatc gagaagtacg
     2221 gaatccccaa cgacatcatc atcgagctgg ccagagagaa caacagcaag gacaaacaga
     2281 agttcatcaa cgaactgcag cgcaagaatg agaagacacg gcagagaatc aacgagatca
     2341 tcggggaata cggcaaccag aacgccaagc ggctggttga aaagattaag ctgcacgacg
     2401 agcaggaggg aaagtgcctg tacagcctgg aaagcatccc actggaagat ttgctgaata
     2461 accccaacta ctacgaggtg gaccacatca tccctagatc tgtgagcttt gacaatagct
     2521 atcagaacaa agtgctggtg aaacagacag agaacagcaa gaagggcaac agaacacctt
     2581 accaatatct gaacagcgga gaggccaaaa tcagctacaa ccagttcaag cagcacatcc
     2641 tgaatctgag caagtccaag gaccggatca gcaagaagaa gaaggagtac ctgctggagg
     2701 agcgggacat caacaagttc gaagtgaaga aggaattcat aaatagaaac ctggtggaca
     2761 cccggtacgc caccagagag cttagcaatt acctgaaagc ctacttcagc gccaacgaca
     2821 tgaacgtgaa gatcaagacc atcaacggct cttttaccga ctacctgagg aaggtgtgga
     2881 agttcaaaaa ggaacggaac cacggctaca agcaccacgc cgaggacgcc ctgatcatcg
     2941 ccaatgccga cttcctgttt aaggagaata agaagctgaa ggaggctaac aaggtgctgg
     3001 agaaacctac cgaaagcaat gaactgaaca aggtcgagaa ctctacacgg atcaagaacg
     3061 aagatgaata caaggaactg ttcaacatcc cccagcaggt gaccgatatc aaggagttca
     3121 aagacttcaa gttcagccac cgggtggata aaaaaccaaa cagacagctc atcaacgaca
     3181 ccctgtactc caccagacag gttggagatg atgcttatat cgtgcagacc atcaagaata
     3241 tctattctaa agataacacc gacctgaaga agcacttcaa caagaaccct gagaaatttc
     3301 tgatgtacca gcatgatcca aagacctttg agaagctgga aaccatcatg aagcagtact
     3361 ccaacgaaaa gaaccccctg gctaaatacc acgaggaaac cggcgaatac ctgaccaagt
     3421 acagcaagaa ggacaacggc cctatcgtga agacaattaa aatgctgtcc aacaaggtcg
     3481 gcaatcacct ggacgtgaca cacaagtacg aggacagcaa gaagcagctg gtgaggctga
     3541 gcatcaagag cctgcggttc gacgtttacc tgaccgacaa gggctacaag ttcattacta
     3601 tctcctacct ggatattctg aagaaggaca gctactacta cattcctaca gagacataca
     3661 acaacttaaa ggccaacaaa tctatcgaca aaagcagcca gttcatcgga tctttctact
     3721 ataacgatct catcgagctg gatggcgaag tgtacaagat cattggagtg aactctgata
     3781 agagaaacct gatcgagctg gacctgccca acatcagata caaggagtac tgcgagctga
     3841 ataacgtgaa gggcgaagcc agaatcaaga agaccattgg caagaaagtc aagtccatca
     3901 aaatcctgca caccgacgtg ctgggaaata tctactacca gaaaaacatc cagaagcctc
     3961 aactgctgtt taagaggggc aacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa
     4021 aaaagaaaaa gggatcctac ccatacgatg ttccagatta cgcttaccca tacgatgttc
     4081 cagattacgc ttacccatac gatgttccag attacgctta agaattccta gagctcgctg
     4141 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4201 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4261 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4321 gggggaggat tgggaagaga atagcaggca tgctggggag gtaccgaggg cctatttccc
     4381 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     4441 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     4501 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     4561 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac
     4621 cacggcaggt ctcagtttta gtactctgga aacagaatct actaaaacaa ggcaaaatgc
     4681 cgtgtttatc tcgtcaactt gttggcgaga tttttgcggc cgcaggaacc cctagtgatg
     4741 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4801 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4861 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4921 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4981 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     5041 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5101 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5161 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5221 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     5281 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     5341 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5401 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5461 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5521 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5581 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5641 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5701 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5761 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5821 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5881 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5941 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6001 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6061 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6121 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6181 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6241 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6301 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6361 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6421 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6481 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6541 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6601 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6661 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6721 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6781 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6841 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6901 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6961 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7021 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7081 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7141 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7201 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7261 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7321 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7381 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7441 gctggccttt tgctcacatg t
//
