5’ TTCTAATACGACTCACTATAGGCATCCTCGGCACCGTCACCCGTTTTAGAGCTAGA 3’
The final sequence of the control sgRNA synthesized using the kit would be:
The sgRNA synthesized from the control oligo targets pBR322 (NEB #N3033, bases 112-131). The complete plasmid sequence can be found here.
Incubating the control sgRNA complexed to S. pyogenes Cas9 in the presence of PvuII-linearized pBR322 plasmid as the target (4361 bp) results in cleavage products of 2423 and 1938 bp (see Figure 6, manual).
Alternatively, uncut (supercoiled) plasmid can be used as a template. On an agarose gel compared to uncut template, the plasmid cut by Cas9 will migrate slower (run higher) than the supercoiled plasmid.
The target sequence for the control sgRNA is: 5’ CATCCTCGGCACCGTCACCC 3’
This sequence is within the tetR(C) gene on the pBR322 plasmid and may be present in other cloning vectors, though the exact sequence in the target should be verified as tetR may be in the opposite orientation or may contain the sequence of another class of tetR genes.