Where is the SalI-XhoI restriction site in the pBluescriptR vector?
This was a hybrid restriction site created during the cloning process (Cap-trapper) for the library creation. Neither of these restriction sites will work to digest the insert from the vector. According to the IMAGE consortium the SalI-XhoI (gtcgag) is located at position 742 of the polylinker sequence (http: //mgc. nci. nih. gov/Vectors/vec_pbluescriptr). Customers can use BamHI (5') and EcoRI (3') to digest out the insert. Other options for sub-cloning are either to use different restriction enzymes lying outside BamHI and EcoRI or to design insert-specific primers based on the insert sequence. You can also find a reference for the cap trapper method here: Carninci et. al. , DNA RESEARCH 4, 61-66 (1997), High Efficiency Selection of Full-length cDNA by Improved Biotinylated Cap Trapper