LOCUS Exported 1060 bp ds-DNA linear SYN 26-MAY-2021 DEFINITION This plasmid contains expression cassette for NmCas9 with N and C NLS and an HA tag, a cassette for expression of tracrRNA, a cassette for cloning crRNA under the control of U6 promoter.. ACCESSION . VERSION . KEYWORDS pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 1060) AUTHORS Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA TITLE Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. JOURNAL Proc Natl Acad Sci U S A. 2013 Aug 12. PUBMED 23940360 REFERENCE 2 (bases 1 to 1060) AUTHORS . TITLE Direct Submission JOURNAL Exported May 26, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..1060 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(205..309) /gene="bla" /label=AmpR promoter promoter complement(384..624) /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind complement(604..624) /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 662..680 /label=T7 Term /note="T7 terminator, reverse primer" promoter complement(699..717) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" misc_RNA complement(759..851) /label=Nm tracrRNA /note="trans-activating CRISPR RNA for the Neisseria meningitidis CRISPR/Cas9 system (Hou et al., 2013)" ORIGIN 1 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 61 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 121 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 181 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 241 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 301 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gtttaaacaa aaaaatgaga 361 cgtggaatcg tctccggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa 421 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc 481 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt 541 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga 601 aggaatcatg ggaaataggc cctcttcctg cccgacctta gatccgaatc catatcgata 661 tgctagttat tgctcagcgg tggcagcagc caactcagcc tatagtgagt cgtattaatt 721 tcgcgggatg tttaaacgcg gccgcaaaaa aaccgaaata aacgatgccc cttaaagcag 781 aagctttaag gggcagagcg ttgcggcaca tcttttcaga cggccttatt gtagcaacgg 841 ttctcatttc ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact 901 ttcaagttac ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact 961 acccaagaaa ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag 1021 tcaaattaat tctaattatc tctctaacag ccttgtatcg //