To increase the chances that the restriction cloning tool will successfully identify the sites you want to use, it is best to create restriction enzyme subsets for your commonly used cloning vectors. You only need to create these lists the first time you perform a restriction cloning operation.
To create a Restriction enzyme subset do the following.
1. Select the pET26B document provided with this tutorial (selecting any sequence will also work).
2. Go Cloning → Find Restriction sites. This will pop out the Restriction Cloning tab associated with the sequence viewer panel.
3. Set Candidate enzymes: to commercially available enzymes.
4. Click on the Advanced button to bring up the list of all enzymes in the commercially available enzymes set.
The pET26B multiple cloning site (MCS) provides the following 11 restriction sites for cloning operations: BamHI, EagI, EcoRI, HindIII, MscI, NcoI, NdeI, NotI, SalI, SacI (SstI) and XhoI. We will now create a restriction enzyme subset that comprises only these sites.
5. Click the check box at the top of the list to deselect all enzymes in the list, then find and select the 11 enzymes for our subset. Click Save Selected Enzymes and name the list pET26B MCS RE sites.
Click OK, the Done. This will create a new Enzyme Set file 
that can be used for all future restriction cloning operations using
the pET26B vector. All files of
this type are indexed so they can be used from anywhere in your Geneious database regardless of the folder they are located in.
Click the link below to go to Part 2: Performing Restriction Cloning.
Part 2: Performing Restriction Cloning
Part 3: Confirming CDS in-frame fusion
Part 4: Troubleshooting