primer Question pVax1 RE / Kozak - (Jun/06/2008 )

I am cloning into pVax1 and have limited experience with Restriction enzymes and kozak seq. so I was looking for some help.

I know that I want to use Nhe1 and BamH for my cosensus however I am not sure how to design the kozak seq. I know there are a few different ways but I could use some help.

Nhe1..........BamHI....................................My insert
GCTAGC-----GGATCC -----(kozak)------- ATGACTAATGCAGCT


As for the Reverse I am using Pme1 & Apa1 and I have to incorporate a stop

We use GCCGCCACC for expression in mammalian cells. This works well for us.