pAAV-RSV-SpCas9

LOCUS       pAAV-RSV-SpCas9                7393 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Express SpCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-RSV-SpCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7393)
  AUTHORS   Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H
  TITLE     The CRISPR/Cas9-created MDM2 T309G enhances vitreous-induced 
            expression of MDM2 and proliferation and survival of cells.
  JOURNAL   J Biol Chem. 2016 May 31. pii: jbc.M116.729467.
  PUBMED    27246850
REFERENCE   2  (bases 1 to 7393)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7393
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        156..381
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     CDS             397..423
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             427..447
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             457..4557
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4561..4581
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4600..4648
                     /note="synthetic polyadenylation signal"
     repeat_region   4656..4796
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4656..4785
                     /label=AAV2 ITR
     rep_origin      4871..5326
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4958..4977)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5168..5189
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5343..5362)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5462..5484
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5522..5540)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5608..5712
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5713..6573
                     /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(5931..5950)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6744..7332
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7233..7252
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agaaatgtag tcttatgcaa tactcttgta
      181 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
      241 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
      301 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
      361 gtgcctagct cgatacataa aaccggtgcc accatgtacc catacgatgt tccagattac
      421 gcttcgccga agaaaaagcg caaggtcgaa gcgtccgaca agaagtacag catcggcctg
      481 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
      541 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
      601 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
      661 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
      721 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
      781 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
      841 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
      901 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
      961 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     1021 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     1081 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     1141 cccggcgaga agaagaatgg cctgttcggc aacctgattg ccctgagcct gggcctgacc
     1201 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     1261 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     1321 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     1381 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     1441 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     1501 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     1561 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     1621 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     1681 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     1741 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     1801 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     1861 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     1921 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     1981 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     2041 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     2101 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     2161 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     2221 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     2281 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     2341 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     2401 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     2461 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     2521 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     2581 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     2641 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     2701 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     2761 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     2821 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     2881 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     2941 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctgaaggac
     3001 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     3061 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     3121 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     3181 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     3241 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     3301 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     3361 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcctacctg
     3421 aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag cgagttcgtg
     3481 tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc
     3541 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     3601 attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa cggcgaaacc
     3661 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     3721 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     3781 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     3841 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     3901 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     3961 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     4021 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     4081 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     4141 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     4201 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     4261 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     4321 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     4381 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     4441 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     4501 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacagc
     4561 cccaagaaga agagaaaggt ggaggccagc taagaattca ataaaagatc tttattttca
     4621 ttagatctgt gtgttggttt tttgtgtgcg gccgcaggaa cccctagtga tggagttggc
     4681 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4741 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4801 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4861 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4921 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4981 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5041 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5101 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5161 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     5221 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5281 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5341 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5401 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5461 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5521 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5581 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5641 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5701 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5761 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5821 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5881 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5941 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6001 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6061 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6121 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6181 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6241 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6301 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6361 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6421 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6481 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6541 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6601 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6661 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6721 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6781 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6841 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6901 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6961 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7021 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7081 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7141 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7201 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7261 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7321 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7381 tttgctcaca tgt
//