I currently have a gene coding for a two domain protein cloned into an expression vector (pET28b). I would like to express the two domains as separate genes but in the same vector.
I though that I could do it by PCR using two primers, one 5′-3′ that anneals with the start of the second domain and that contains a LIC compatible 5′ sequence. The other 3′-5′ primer anneals with the end of the first domain and also contains a LIC compatible sequence on its 3′ end. I will obtain then a linearized vector that, after treatment with T4 Pol+dATP I can anneal with a pETDuet LIC adaptor that will provide the T7 promoter, RBS, etc. for the expression of the second domain.
As I am not a molecular biology expert I will appreciate the opinion of the forum about if this strategy is a reasonable one to try.