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[cancerous]

Dear experts,

I have cloned my gene of interest into pMSCV-PIG vector. The brief insert sequence is as followed;

CTCGAG(= XhoI) GCCACC (=Kozak) ATG-------TAA (ORF) GTTAAC (=HpaI)

I used two enzyme sites for this cloning (XhoI and HpaI). In transient transfection in 293T cells, I could not get any overexpression.

I haven't used pMSCV-PIG vector before, so don't know if inserting ORF without extra mRNA sequence at 5' and 3' ends is enough.

Could anyone tell me any suggestion?

Thanks.

Cancerous

[bob1]

It should be fine to insert without any UTRs.

What times have you tried to detect the protein at following transfection?  Have you looked to see if the mRNA is expressed?