Four base mutations are introduced to the C-terminal of endogenous Vmat to introduce a stop codon, mutate the guide PAM, and introduce a silent BglII site.
T13518135A
T?A
L?term
A nonsense mutation is introduced at L589 of Vmat-RC, RE, and RF, which also creates a BglII site. There is also a mutation in the PAM site (ATG to TCA) starting six bases downstream.