I am using the clontech adenoX in fusion (system 3) to generate an shRNA adenovirus due to the apparent simplicity of the protocol (no shuttle vector cloning required). For this approach, you simply amplify the shRNA cassette (designed using their software) with primers containing 15bp overhangs that overlap with the ends of the linearized target adenoX infusion vector. What I'm trying to do is amplify the human U6 promoter from genomic DNA (which seems to be good for shRNA expression) and then fuse it to the shRNA cassette that I designed. My only confusion is how to design the overlap between the end of the U6 promoter and the beginning of the shRNA cassette. I'm not sure really how pol-III initiates transcription of small RNAs, so I don't know if I should have a gap sequence between the last few nucleotides of the U6 promoter and the shRNA cassette. Any clarification would be appreciated.

The idea for the primers is:

vector------- (5')

               x

             -----U6 promoter---------

                                              ?

                                            -----shRNA cassette--------

                                                                                 x

                                                                               -----3' Advector