Gateway cloning with 3 entry vectors - (Aug/21/2007 )

Hi,

I'm new to the Gateway cloning system, but have a project that requires me to recombine 3 entry vectors into a Gateway destination vector. My question is do I need 3 different entry vectors for the destination cloning or can I use the same vector with 3 different constructs?

I have pENTR with my gene of interest.
I have to remove a promoter from a Gateway-incompatible vector & clone it into a Gateway-compatible entry vector. Can I use pENTR?
I have a GFP/stop codon construct in a Gateway-compatible entry vector (not sure which entry vector).

Many thanks for your input.

You could clone all the different peices into pENTR vector and then do gateway cloning. Use the pENTR vector like a cloning vector for assembling different parts of the final expression cassette.

Thanks Scolix.

I think my confusion lies in the recombination sites of the entry vector. For example, I've got my gene of interest in pENTR, which has attL1 and attL2 sites. Do I need a different pENTR that has attL4/attR1 and attR2/attL3 for my 5' and 3' elements, respectively?

Thanks again.

use the same pENTR with the transgene and clone other fragments into it. I dont think you could use 3 different pENTR vectors and clone a different fragment from each of them into a single destination vector. Ask the invitrogen support. may be they could suggest.

Thanks for your suggestions!