For our agrobacterium mediated plant transformation study, we cloned our gene of interest into a binary T DNA vector called pGFPGUSPlus. As the vector construction strategy, in the pGFPGUSPlus vector, we briefly replaced the GUS (Beta-Glucuronidase) ORF with the ORF of our gene of interest using the standard cloning procedures. We therefore aimed to have our gene of interest expressed under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S Promoter).

However we later have realized that the kozak sequence belonging to the GUS gene was remained in the reconstructed vector. In this final case, the construct includes: 5’ CaMV 35S Promoter – Kozak Sequence of the GUS Gene – Kozak Sequence of Our Gene of Interest –  ORF of Our Gene of Interest 3’ respectively.     

The AUG start site in the upstream kozak sequence is not in frame with the AUG start site of our protein of interest. Thus, in this situation, would the expression of the protein encoded by our gene of interest be affected by the additional kozak sequence located upstream of our actual kozak sequence?

Any comment on this issue will be very helpful for us.

Thanks