General musings from the MacVector team about sequence analysis, molecular biology, the Mac in general and of course your favorite sequence analysis app for the Mac!

How to find one-out Restriction Sites that will change a protein coding sequence

If you are interested in making changes to a protein sequence, it is often useful to make a change to the coding DNA that will create a restriction enzyme site. Conversely, there may be times that you would like to create a site without affecting a protein coding region. You can accomplish this using the Analyze | Quicktest Primer (Individual) function.

  • Make sure that any protein coding regions in your target DNA are annotated as CDS features – this is critical as MacVector will key off such features when displaying translations.

  • Select a ~100nt region where you want to search for sites and then choose Analyze | Quicktest Primer…

The resulting dialog shows the selected sequence aligned to the parent molecule, complete with restriction sites and the translations of the protein coding features (here shown in blue as they are on the minus strand).

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Restriction sites in black are those currently present in the sequence. Those in red and green are “one-out” sites (e.g. a 3 out of 4 or 5 out of 6 match), where changing a single residue would create a new site. Those in red would change the amino acid sequence of the encoded protein. Those in green would be silent changes, not affecting the protein sequence. If you click and hold on a restriction enzyme site, the sequence is temporarily changed to match the restriction site and any changes are reflected above the line. In the example above, clicking and holding on a red BamHI* site would change a Glu residue to an Asp in the protein coding sequence, and would create BspEI and DpnI sites in addition to a BamHI site in the molecule. If you double-click on a site, the change is entered permanently. You can also nudge the sequence to the left and right using the arrow keys. If your display does not look similar to the example, click on the Settings button and make sure you have “Include one-out sites” and “Use all enzymes” selected.

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