0 zhang lab plasmid for expressing a chimeric guide rna. Plus a puromycin resistance marker and human codon. A previous colleague designed the target sequence inserts for px. Using overhangs that don. T match the overhangs suggested in fig. 4 of the ran fa et al.
The guide sequence is cloned into this plasmid using bbsi sites. Can be used to confirm the grna sequence after cloning into the plasmid. Please see the cloning protocol provided by the zhang lab for more details. Empty grna expression vectors for insertion of custom grna target sequences.
There is just one bbsi cloning site for 1 grna sequence in this vector. S lab contains the insert hspcas9. Puro and is published in nat protoc.
Thermo scientific bpii. Restriction enzyme recognizes gaagac. Sites and cuts best at 37. Bbsi, bpuai, bstv2i. See reaction conditions for restriction enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes. This protocol describes how to clone protospacer adapters into the crispr plasmids px.