Add a Restriction Site to a Coding Sequence Without Changing the Translation
How do I add an enzyme site to a translated sequence and ensure the translation is not changed?
Open the Target Sequence
Open a file with the target translated feature.
Select the Target Translated Feature
In Map or Sequence view, click on the targeted translated feature to select it.
Start the "Silent Mutation:Add Enzyme Site" Tool
Click Enzymes → Silent Mutations → Add New Enzyme Sites in Selected Translation....
Set the Criteria for New Site Identification
The "Add a Restriction site" tool may find many potential sites on your sequence. Use the options on the left of the "Add New Enzyme Sites" window to limit the number of potential sites that are found.
- Select an "Enzyme Set" with sites to be considered for addition to the sequence. Choose from the default sets, or use a custom set. See the Guide chapter Save or Export Enzyme Set to learn more about how to create a custom enzyme set.
- Set the "Existing sites for the same enzyme" limits to consider or exclude sites that are already be present in the entire sequence, or in the selected translation.
- Set the range allowed for the enzyme recognition sequence length.
- Set the allowed number of base changes to create the new site.
Choose a New Site
The list of potential "new sites" is based on the criteria set in the left hand panel and shows all potential new sites that can be added to the sequence without altering the translation. More than one new site for any enzyme may be able to be created.
Click on an Enzyme name in the list (1) to view the location of the potential new site in the sequence (2) and see the nucleotide changes required to create the select site (3, highlighted red). The location of the selected potential new site with respect to the target translated feature is shown as a vertical bar in the minimap (4).
If site position within the CDS feature is important, click on the"New Site" column header to order the list of new potential sites based on position in the sequence.
Select One or More Sites for Addition to the Sequence
Check the box next to one or more enzyme sites to select them for addition to the sequence.
Choose How to Add the Selected Site(s)
Click the dropdown menu to choose how to generate or annotate the mutation required to add the enzyme site(s).
Expand each of the following article sections to learn more about the different options.
Select the option "Mutate the sequence" to add the new site to the sequence. Click Add x Enzyme Site(s).
The sequence will be mutated to add the selected site(s) using alternative synonymous codons .
Switch to Sequence view, and turn on History Colors via the side toolbar to highlight (in red) the nucleotide change[s] made to create the site(s).
Click Save to saved the edited file.
In the History view, the changes will be recorded as a "Silent Mutagenesis" step, and will note the enzyme site added to the sequence.
Click the dropdown and choose "Default parameters for New Enzyme Sites...".
If required, change the default settings for the design of the mutagenic primers.
Select the option "Mutate the sequence with mutagenic primers".
Set the bacterial transformation strain (optional).
Give the mutagenized product sequence a name.
Click Add X Enzyme Site(s) to create a new product sequence. SnapGene will automatically design a pair of primers suitable for PCR-based mutagenesis, and simulate PCR and recircularization of the product to introduce the changed codon(s), and add the new restriction site(s).
In the History view, the change will be recorded as a "Silent Mutagenesis" step, and will note the enzyme site added, and the primers designed to add the enzyme site.
Choose "Annotate mutation with a feature" and click Add X Enzyme Sites(s) to add a feature to the sequence.
A new feature of Type: misc_difference will be added to the sequence and named based on the potential new enzyme site. Hover over the Feature to view the /note qualifier detailing the change(s) to the sequence required to generate the new enzyme site.
Note: The "Annotate mutation with a feature" option DOES NOT perform a mutagenesis step and the enzyme site is not added to the sequence. No sequence change is recorded in History.
Choose "Annotate mutation with a feature and mutagenic primers" and click Add X Enzyme site(s) to add a feature to the sequence and to design mutagenic primers.
New mutagenic primers are added to the sequence, but no change is made to the DNA sequence. The primers for mutagenesis will show a mismatch at the nucleotide position(s) targeted for "silent mutagenesis".
A new feature of Type: misc_difference is also added to the sequence, named for the potential new site. Hover over the Feature to view the /note qualifier detailing the change(s) to the sequence required to generate the new enzyme site.
Click File → Save to save the feature and primer annotations on the sequence.
Note: The "Annotate mutation with a feature and mutagenic primers" option DOES NOT perform a mutagenesis step and the enzyme site is not added to the sequence.
Choose "Export a list of the chosen new sites" to export a list of all selected new sites.
Choose "Export a list of the possible new sites" to export a list of all possible new sites.
Click Export List to export the list to a PDF document.
View the PDF document to see the list of potential new sites.