5´- NAGA -3´ PAM sequence allows targeting of additional genomic regions
Ideal for direct introduction of Cas9/sgRNA complexes
Compatible with the EnGen Mutation Detection Kit (NEB #E3321S)
Active in in vitro reactions from 20°C to 45°C
Product Information
EnGen Seq1 Cas9 from Streptococcus equinus is an RNA-guided DNA endonuclease that catalyzes site-specific cleavage of double-stranded DNA (dsDNA). Targeting requires the formation of a ribonucleoprotein complex of Cas9 and a 116-nucleotide single guide RNA (sgRNA). The sgRNA encodes a 20-nucleotide sequence with complementarity to the DNA target strand immediately upstream of a 5´– NAGA –3´ protospacer adjacent motif (PAM) on the non-target strand. DNA cleavage by EnGen Seq1 Cas9 produces a double-stranded break occurring 3 nucleotides upstream of the PAM. EnGen Seq1 Cas9 has Simian virus 40 (SV40) T antigen nuclear localization signals (NLS) at both the N- and C-termini of the protein.
Figure 1. Schematic representation of S. equinus Cas9 nuclease complexed with a single guide RNA and target DNA
S. equinus Cas9 (Seq1 Cas9) protein is shown in beige, single guide RNA (sgRNA) is depicted in blue, and the DNA is shown in grey, green, and red. The DNA target, or protospacer, is shown in green and the base pairing complementarity of the target strand with the guide RNA in the R-loop is illustrated. The protospacer adjacent motif (PAM) which is required for Cas9 binding to DNA is shown in red and the locations of DNA strand cleavage are indicated with black triangles.
Product Source
An E. coli strain that carries the cloned Cas9 gene from Streptococcus equinus with N- and C-terminal Simian virus 40 (SV40) T antigen nuclear localization signal (NLS) and a C-terminal 6XHis tag.
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