You wish to use traditional (PCR and restriction enzyme-based) cloning techniques to clone your gene of interest (gene 3021) into the plasmid vector pBPS (to make the recombinant plasmid pBPS-3021).  You wish to express the gene product as a fusion protein in E. coli. 

For each of the plasmid features (A-E), as shown on the diagram above, answer the following questions:

A.  What is the purpose of the promoter and does it need to be before (upstream of) the MCS as shown in the diagram?

B.  The gene of interest will be inserted into the MCS, briefly explain how this will be achieved.

C.  Name one "tag" or fusion protein commonly used in plasmid expression vectors, and, based on the figure provided, briefly explain whether or not you will need to insert the 3021 gene in-frame with this tag.

D.  What is the purpose of the origin of replication?  Do high-copy plasmids have multiple ori's?

E.  Name one selectable marker commonly used in plasmid expression vectors and briefly explain its purpose.

(2 marks each)