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[intandiana]

Hi all,

I have a problem to express bacterial gene into pichia expression system. By referring to the manual, to express gene into pPICZ (no signal peptide), I need to include yeast consensus sequence (Kozak sequence) to the gene which is be done during primer synthesis.

So, is it mean that i need to include these below in my primer design?
1) RE site (EcoRI): GAACTC
2) Kozak sequence: G/ANNATGG
3) about 20 first nucleotide gene (exclude ATG)

so it could be like this: GAACTC-ACCATGG-20bp of nucleotide gene. However, the G, may shift the protein translation frame of my gene. If i just ignore the G (means exclude it), is my primer may work out for expression into pPICZ vector of Pichia?

please help me...