Identify the restriction sites of pBR322, where if genes are inserted results in the phenomenon insertional inactivation.

I. Pvu I            II. Pvu II            III. Sal I               IV. Pst I

The restriction sites Pvu I and Pst I are present within the marker gene ${\mathrm{amp}}^{\mathrm{R}}$and Sal I within another marker gene ${\mathrm{tet}}^{\mathrm{R}}$. If genes are inserted at these places it results in insertional inactivation of marker genes. Since Pvu II is not present within marker genes, if a gene is inserted at this site it does not result in inactivation of expression of marker genes.