How to add a lethal gene to my plasmid for cloning selection? - (Apr/14/2007 )

My vector is for Eukaryotic expression.
Any suggestion will be great helpful.
Thank you!

-Hyland-

cut n paste ?

Put your lethel gene under an inducible promoter which has very low auto induction background?

-perneseblue-

QUOTE (perneseblue @ Apr 20 2007, 02:53 PM)

cut n paste ?

Put your lethel gene under an inducible promoter which has very low auto induction background?

I want to make a construct which contains a lethal gene (cause cell death). ligation of a PCR fragment will disrupt the expression of the lethal gene permitting groth of only recombiants upon transfornation. But the lethal gene don`t contain any restriction sites. If I fuse the lethal gene to the MCS, there will be a long noncoding domain between the promoter and the insertion or instertion between the PolyA. I am afraid that will affect the expression of my insertion.
Thank you for any suggestion!

-Hyland-

Is it not possible to PCR your gene using primers that introduce a unique restriction sites, sites which would allow you to ligate your gene close to your promoter. And if you promoter does not have restriction site that you could use, why not PCR amply your promoter as well?

-perneseblue-