In my last post I described how you can quickly create and annotate features onto a DNA sequence, although the post was primarily aimed at users who are new to MacVector. In this post I’ll take a look at how you can quickly and easily annotate a DNA sequence with features based on the results of a MacVector analysis. For example, we’ll see that you can run an Open Reading Frame search, then click on the graphical object representing a found ORF, and annotate that as a CDS feature.
Most of these workflows require you to right-click in a window to bring up a context-sensitive menu. If you don’t have a two button mouse, you can accomplish the same thing by holding down the ctrl key and clicking the mouse button.
Restriction Enzyme Sites: After running a restriction enzyme analysis (Analyze | Restriction Enzyme Analysis…), you can right-click on a site in the Cut Site Map to automatically create a misc_feature feature at the cut site;
A new feature is automatically created and displayed at the appropriate location;
And can also be seen in the Features tab;
While misc_feature is really the best feature type you should use for indicating a restriction enzyme cut site, there may be times when you would prefer to use a different feature type. If you hold down the option key, the menu item changes from “Create misc_feature Feature” to simply “Create Feature…“;
After selecting the Create Feature… menu item, the normal Feature Editor pane appears, pre-filled with the location information and a suitable description, but now offering you the opportunity to change the feature type, or any other information you would like to have associated with the feature;
Subsequence Search Matches: Very similar to restriction enzyme searches, you can annotate the results of subsequence searches, by default as misc_feature features. Unlike restriction enzyme searches, which return a specific single cut site, subsequence searches return the start and stop location where the subsequence matches, so the corresponding feature that is created has both a start and a stop location, and is also strand specific. The features are created using a Hollow Arrow feature type to indicate the start, stop and strand information (e.g. when the match is to the minus strand the arrow will point in the opposite direction). Here’s an example from a subsequence search of an .nsub file containing common sequencing primers against pUC19. In this case, I used the option key approach described above to set the feature type to primer_bind rather than the default misc_feature;
Open Reading Frames: If you use the Analyze | Open Reading Frames menu option to find open reading frames on a DNA sequence, you can click on an ORF result feature to create a new CDS feature;
Primers and Predicted PCR Products: From the Analyze | Primers | Primer3 analysis results Map window, you can right-click on either an individual primer (orange in the image below) or on a predicted product (purple);
Now the great thing about all these feature creation functions is that they always generate a totally GenBank compliant annotation. MacVector really does a good job on adhering to the GenBank standard for sequences and annotations. If you ever want to save a sequence in GenBank format, select the File | Save As… menu option then choose GenBank Text Format from the Format menu. Now, you can also save sequence files in a format ready for use by the NCBI Sequin program, but maybe thats a post for another day.
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