Transfering a DNA construct from one vector to another - in frame/not in frame? (Nov/29/2005 )

Hi all,

I have a question concerning the cloning of a gene construct into a constitutive mammalian expression vector.

I currently have a DNA construct inserted in a pcDNA3 vector and I would to transfer it to another expression vector such as pcDNA6 for instance. The problem is that I don't know the exact sequence of my insert, meaning I have no clue about the UTR sequences that have been included inside the plasmid when it has been made at first. So, how can I be sure that the gene is going to be in frame with the CMV promoter and my protein expressed ? I know what restriction enzymes they used to insert the construct into the pcDNA3 vector. Can it be helpful for me, meaning if I can use the same restriction enzymes to insert into pcDNA6, can I be sure that my gene will be in frame?

Also, the pcDNA6 has a myc-His tag, but the guys who made the constructs already included a HA-flag tag inside the pcDNA3 plasmid. Do you think it is going to be a problem for the expression of the protein to have 4 tags at the end ??!

I hope I'm clear... and not too confusing...

Thank you for any suggestions you would have !

M-kun

hi
as you know there is tags at the end of your plasmid you can easyly design primers to sequence your fragment til the 3'. A bit long but seems necessary in your case.

4tags are not a problem. Colleagues are working with 6tag... so 4 is ok according to me

Ok. Thank you for the tip fred_33, I appreciate.

Merci du coup de main !

M-kun