Cloning and expression problem - (Sep/24/2013 )

I try to clone a gene, simply cut and paste it into another plasmid. I took the gene out with restriction enzymes blunt from one size and sticky from another site and inserted into blunt/sticky ends in the MCS of another expression vector. I've got positive clones, The DNA is ok, I can cut out the insert, I also sequenced the insert and it was ok. However when I transfect it into mammalian cell line, I don't see the expression. What could be a problem?

Thank you

I have the same problem with a retroviral vector! I am breaking my head. The same insert expressed from another vector. It happened to my colleague and finally he recloned the insert into another vector and it worked....

Thank you! I thought that this is the problem but my PI talked about wrong plan of cloning, She said that I cloned it into a wrong frame that's why the promoter do not work properly. As far as a know promoter works in all reading frames and that is not a problem. Isn't it?

Protein expression and ORF are determined by ATG and Kozac sequence not by the promoter (that is where transcription starts). Do you have Kozac sequence in your vector? BTW-I have it but my protein still does not express....

I use pcDNA3.1 vector for cloning.I didn't check it but I suppose that a commercial vector have it. I will re-check the sequence. Thank you!!!!
 

But the Kozac has to go directly in front of your ATG. You should add it.