Editing Pichia EXpression (section)

== Cloning pJ912-19<br>  ==

Clone gene of interest into the '''Xho1''' and one of these sites: Nhe1, Not1, Spe1 

For PCR cloning: 

Forward primer: nnnnnn '''CTC GAG''' aaa agg ---Gene specific 20n<br>Rev primer use Nhe1, Not1, Spe1 with stop codon. After clone verification by PCR or digestion, use one of the following enzymes to cut plasmid<br> 

'''SacI''', SwaI, BamHI<br> *Zeocin selection in E.coli and Pichia pastoris<br>*AOX1 promoter for methanol inducible expression of gene of interest in Pichia pastoris<br>*Alpha factor signal sequence for secreted expression of gene of interest<br>*For integration at AOX1 locus, linearization with SwaI or '''SacI'''<br> +Choose SwaI to linearize vector for low to medium expression level (random integration);<br> +Choose '''SacI '''to linearize vector for increased expression<br>*For integration at AOX1 Terminator, linearization with '''BamHI'''<br>*To remove E. coli sequence from the integrated expression cassette, linearization with SwaI (or SacI) and PstI<br>*'''Your gene must NOT include BamHI, BsaI, PstI, SacI or SwaI sites '''<br> 

Purify linearizd plasmid DNA using spin column, eluted with '''hot water (&gt;65 oC)'''.<br>