Restriction cloning in Geneious requires you to have an insert which is already digested, OR an insert with two restriction site annotations on it. The cloning tool will then digest the sequence at the annotated sites, discard the ends and insert the fragment into the vector. In the next part of this tutorial we will produce an insert with two NcoI annotations on it.
Open the Human Proinsulin sequence.
Using Find Restriction Sites you're going to locate the existing NcoI site. This time select Cut Anywhere, then click the Advanced button to bring up the list of enzymes. Scroll down to find NcoI and select just that one then click Done.
This will annotate just one NcoI site near the beginning of the CDS. Click Apply and save the document.
Next we are going to introduce a cut site at the 3' end of the gene using PCR.