To introduce restriction sites for primers:

  1. Create or import primers or primer pairs.
  1. From the Primer Design view, either click on the Introduce Restriction Site tool ( ) at the top left of the window, or choose Priming > Introduce Restriction Site.
  1. Click between the residues where the restriction site should be added. This opens a top level menu with the restriction enzymes identified to cleave the sequence at a specific location:
    • <Specific enzymes by name>

    • Non-silent substitutions

    • Any enzyme – Select this option to choose an enzyme from a submenu. If you are looking for a particular enzyme, click on the top-level menu, then type the first 1-2 letters of that enzyme. This will automatically navigate to the part of the submenu list where the enzyme is located.

    • Enzymes that don’t cut product – See note for Any enzyme, above.

    • Introduce a stop

New restriction sites are displayed in magenta, as well as nucleotides in the primer sequence and their translations that differ from the template. Restriction enzymes whose recognition sites are eliminated by the edit are shown in blue.