71330  pCDF-1b DNA

The pCDF-1b plasmid features coexpression capabilities as well as the ability to express fusion proteins with a N-terminal His•Tag that results in native protein after purification and cleavage. This plasmid carries an origin derived from CloDF13 (1) and streptomycin/spectinomycin resistance, allowing for the option of coexpression with many other Novagen T7-based expression vectors.

The vector contains a T7 promoter, lac operator, ribosome binding site (rbs), an amino terminal His•Tag^® coding sequence, and multiple cloning site (MCS) regions designed to allow the generation of target proteins with minimal vector-encoded fusion. The Pml I cloning site allows direct fusion to the His•Tag sequence for inserts that are blunt and in the appropriate reading frame. For applications requiring a removable amino-terminal His•Tag sequence, the MCS includes a PshA I cloning site (GACNNNNGTC). The PshA I site overlaps the cleavage site for the enterokinase (EK) protease (AspAspAspAspLys). Cloning appropriately designed inserts into this site re-creates the full EK site and allows all amino-terminal vector-encoded sequences to be removed by EK digestion. The remainder of the MCS encodes restriction enzyme sites found in many of other Novagen expression vectors to facilitate insert transfer. An optional C-terminal S•Tag™ coding sequence is compatible with purification, detection, and quantification (2).