LOCUS Exported 8430 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION A human codon-optimized SpCas9 nickase and chimeric guide RNA expression plasmid.. ACCESSION . VERSION . KEYWORDS pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8430) AUTHORS Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F TITLE Multiplex Genome Engineering Using CRISPR/Cas Systems. JOURNAL Science. 2013 Jan 3. PUBMED 23287718 REFERENCE 2 (bases 1 to 8430) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8430 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 120..405 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" misc_RNA complement(502..577) /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter complement(604..844) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(654..673) /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind complement(824..844) /label=hU6-F /note="Human U6 promoter, forward primer" rep_origin complement(906..1494) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(986..1005) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(1665..2525) /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 2288..2307 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(2526..2630) /gene="bla" /label=AmpR promoter primer_bind 2698..2716 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(2754..2776) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 2876..2895 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" rep_origin complement(2912..3367) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3049..3070) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 3261..3280 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 3442..3582 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 3453..3582 /label=AAV2 ITR polyA_signal 3591..3798 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 3787..3804 /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" CDS complement(3841..3861) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(3865..7965) /codon_start=1 /product="nickase mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9(D10A) /note="generates RNA-guided single strand nicks in DNA due to the D10A mutation in the RuvC catalytic domain" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS complement(7975..7995) /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS complement(7999..8025) /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" intron 8046..8273 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" promoter complement(8274..118) /label=chicken beta-actin promoter ORIGIN 1 cccccccccc ccgcccccat cgctgcacaa aataattaaa aaataaataa atacaaaatt 61 gggggtgggg agggggggga gatggggaga gtgaagcaga acgtggggct cacctcgacc 121 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc 181 atgtactggg cacaatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta 241 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc 301 cattgacgtc aatggaaagt ccctattggc gttactattg acgtcaatgg gcgggggtcg 361 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgggtac ctctagagcc 421 atttgtctgc agaattggcg cacgcgctaa aaacggacta gccttatttt aacttgctat 481 ttctagctct aaaacaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac 541 tagccttatt ttaacttgct atttctagct ctaaaacagg tcttctcgaa gacccggtgt 601 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa 661 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt 721 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa 781 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc 841 cctcacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 901 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 961 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1021 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1081 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 1141 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1201 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1261 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1321 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 1381 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 1441 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 1501 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 1561 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 1621 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 1681 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 1741 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 1801 ccgcggcttc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 1861 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 1921 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 1981 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 2041 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 2101 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 2161 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 2221 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 2281 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 2341 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 2401 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 2461 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 2521 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 2581 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 2641 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 2701 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 2761 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2821 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 2881 tcagagcaga ttgtactgag agtgcaccat aaaattgtaa acgttaatat tttgttaaaa 2941 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aatagaccga aatcggcaaa 3001 atcccttata aatcaaaaga atagcccgag atagagttga gtgttgttcc agtttggaac 3061 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 3121 ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt 3181 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 3241 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctaa ggcgctggca 3301 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 3361 ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat accgcacaga tgcgtaagga 3421 gaaaataccg catcaggcgc ccctgcaggc agctgcgcgc tcgctcgctc actgaggccg 3481 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag 3541 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc tccccagcat 3601 gcctgctatt ctcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa 3661 tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag 3721 tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct 3781 ggcaactaga aggcacagtc gaggctgatc agcgagctct aggaattctt agctggcctc 3841 cacctttctc ttcttcttgg ggctgtcgcc tcccagctga gacaggtcga tccgtgtctc 3901 gtacaggccg gtgatgctct ggtggatcag ggtggcgtcc agcacctctt tggtgctggt 3961 gtacctcttc cggtcgatgg tggtgtcaaa gtacttgaag gcggcagggg ctcccagatt 4021 ggtcagggta aacaggtgga tgatattctc ggcctgctct ctgatgggct tatcccggtg 4081 cttgttgtag gcggacagca ctttgtccag attagcgtcg gccaggatca ctctcttgga 4141 gaactcgctg atctgctcga tgatctcgtc caggtagtgc ttgtgctgtt ccacaaacag 4201 ctgtttctgc tcattatcct cgggggagcc cttcagcttc tcatagtggc tggccaggta 4261 caggaagttc acatatttgg agggcagggc cagttcgttt cccttctgca gttcgccggc 4321 agaggccagc attctcttcc ggccgttttc cagctcgaac agggagtact taggcagctt 4381 gatgatcagg tcctttttca cttctttgta gcccttggct tccagaaagt cgatgggatt 4441 cttctcgaag ctgcttcttt ccatgatggt gatccccagc agctctttca cactcttcag 4501 tttcttggac ttgccctttt ccactttggc caccaccagc acagaatagg ccacggtggg 4561 gctgtcgaag ccgccgtact tcttagggtc ccagtccttc tttctggcga tcagcttatc 4621 gctgttcctc ttgggcagga tagactcttt gctgaagccg cctgtctgca cctcggtctt 4681 tttcacgata ttcacttggg gcatgctcag cactttccgc acggtggcaa aatcccggcc 4741 cttatcccac acgatctccc cggtttcgcc gtttgtctcg atcagaggcc gcttccggat 4801 ctcgccgttg gccagggtaa tctcggtctt gaaaaagttc atgatgttgc tgtagaagaa 4861 gtacttggcg gtagccttgc cgatttcctg ctcgctcttg gcgatcatct tccgcacgtc 4921 gtacaccttg tagtcgccgt acacgaactc gctttccagc ttagggtact ttttgatcag 4981 ggcggttccc acgacggcgt tcaggtaggc gtcgtgggcg tggtggtagt tgttgatctc 5041 gcgcactttg taaaactgga aatccttccg gaaatcggac accagcttgg acttcagggt 5101 gatcactttc acttcccgga tcagcttgtc attctcgtcg tacttagtgt tcatccggga 5161 gtccaggatc tgtgccacgt gctttgtgat ctgccgggtt tccaccagct gtctcttgat 5221 gaagccggcc ttatccagtt cgctcaggcc gcctctctcg gccttggtca gattgtcgaa 5281 ctttctctgg gtaatcagct tggcgttcag cagctgccgc cagtagttct tcatcttctt 5341 cacgacctct tcggagggca cgttgtcgct cttgccccgg ttcttgtcgc ttctggtcag 5401 caccttgttg tcgatggagt cgtccttcag aaagctctga ggcacgatat ggtccacatc 5461 gtagtcggac agccggttga tgtccagttc ctggtccacg tacatatccc gcccattctg 5521 caggtagtac aggtacagct tctcgttctg cagctgggtg ttttccacgg ggtgttcttt 5581 caggatctgg ctgcccagct ctttgatgcc ctcttcgatc cgcttcattc tctcgcggct 5641 gttcttctgt cccttctggg tggtctggtt ctctctggcc atttcgatca cgatgttctc 5701 gggcttgtgc cggcccatca ctttcacgag ctcgtccacc accttcactg tctgcaggat 5761 gcccttctta atggcggggc tgccggccag attggcaatg tgctcgtgca ggctatcgcc 5821 ctggccggac acctgggctt tctggatgtc ctctttaaag gtcaggctgt cgtcgtggat 5881 cagctgcatg aagtttctgt tggcgaagcc gtcggacttc aggaaatcca ggattgtctt 5941 gccggactgc ttgtcccgga tgccgttgat cagcttccgg ctcagcctgc cccagccggt 6001 gtatctccgc cgcttcagct gcttcatcac tttgtcgtcg aacaggtggg cataggtttt 6061 cagccgttcc tcgatcatct ctctgtcctc aaacagtgtc agggtcagca cgatatcttc 6121 cagaatgtcc tcgttttcct cattgtccag gaagtccttg tccttgataa ttttcagcag 6181 atcgtggtat gtgcccaggg aggcgttgaa ccgatcttcc acgccggaga tttccacgga 6241 gtcgaagcac tcgattttct tgaagtagtc ctctttcagc tgcttcacgg tcactttccg 6301 gttggtcttg aacagcaggt ccacgatggc ctttttctgc tcgccgctca ggaaggcggg 6361 ctttctcatt ccctcggtca cgtatttcac tttggtcagc tcgttataca cggtgaagta 6421 ctcgtacagc aggctgtgct tgggcagcac cttctcgttg ggcaggttct tatcgaagtt 6481 ggtcatccgc tcgatgaagc tctgggcgga agcgcccttg tccaccactt cctcgaagtt 6541 ccagggggtg atggtttcct cgctctttct ggtcatccag gcgaatctgc tgtttcccct 6601 ggccagaggg cccacgtagt aggggatgcg gaaggtcagg atcttctcga tcttttcccg 6661 gttgtccttc aggaatgggt aaaaatcttc ctgccgccgc agaatggcgt gcagctctcc 6721 caggtggatc tggtggggga tgctgccgtt gtcgaaggtc cgctgcttcc gcagcaggtc 6781 ctctctgttc agcttcacga gcagttcctc ggtgccgtcc atcttttcca ggatgggctt 6841 gatgaacttg tagaactctt cctggctggc tccgccgtca atgtagccgg cgtagccgtt 6901 cttgctctgg tcgaagaaaa tctctttgta cttctcaggc agctgctgcc gcacgagagc 6961 tttcagcagg gtcaggtcct ggtggtgctc gtcgtatctc ttgatcatag aggcgctcag 7021 gggggccttg gtgatctcgg tgttcactct caggatgtcg ctcagcagga tggcgtcgga 7081 caggttcttg gcggccagaa acaggtcggc gtactggtcg ccgatctggg ccagcaggtt 7141 gtccaggtcg tcgtcgtagg tgtccttgct cagctgcagt ttggcatcct cggccaggtc 7201 gaagttgctc ttgaagttgg gggtcaggcc caggctcagg gcaatcaggt tgccgaacag 7261 gccattcttc ttctcgccgg gcagctgggc gatcagattt tccagccgtc tgctcttgct 7321 cagtctggca gacaggatgg ccttggcgtc cacgccgctg gcgttgatgg ggttttcctc 7381 gaacagctgg ttgtaggtct gcaccagctg gatgaacagc ttgtccacgt cgctgttgtc 7441 ggggttcagg tcgccctcga tcaggaagtg gccccggaac ttgatcatgt gggccagggc 7501 cagatagatc agccgcaggt cggccttgtc ggtgctgtcc accagtttct ttctcaggtg 7561 gtagatggtg gggtacttct cgtggtaggc cacctcgtcc acgatgttgc cgaagatggg 7621 gtgccgctcg tgcttcttat cctcttccac caggaaggac tcttccagtc tgtggaagaa 7681 gctgtcgtcc accttggcca tctcgttgct gaagatctct tgcagatagc agatccggtt 7741 cttccgtctg gtgtatcttc ttctggcggt tctcttcagc cgggtggcct cggctgtttc 7801 gccgctgtcg aacagcaggg ctccgatcag gttcttcttg atgctgtgcc ggtcggtgtt 7861 gcccagcacc ttgaatttct tgctgggcac cttgtactcg tcggtgatca cggcccagcc 7921 cacagagttg gtgccgatgg ccaggccgat gctgtacttc ttgtcggacg cttcgacctt 7981 gcgctttttc ttcggcgaag cgtaatctgg aacatcgtat gggtacatgg tggcaccggt 8041 ccaacctgaa aaaaagtgat ttcaggcagg tgctccaggt aattaaacat taatacccca 8101 ccaaccaacc atcccttaaa cccttacctc ttgctcagct aattacagcc cggaggagaa 8161 gggccgtccc gcccgctcac ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg 8221 cgcgaggcgg cggcggagcg gggcacgggg cgaaggcagc gcgcagcgac tcccgcccgc 8281 cgcgcgcttc gctttttata gggccgccgc cgccgccgcc tcgccataaa aggaaacttt 8341 cggagcgcgc cgctctgatt ggctgccgcc gcacctctcc gcctcgcccc gccccgcccc 8401 tcgccccgcc ccgccccgcc tggcgcgcgc //