You can design primers by specifying two flanking regions and selecting left and right primers from these. This is similar to the Amplify Feature/Region method but it is much more flexible, as you can specify more precisely the region that each primer is selected from.
To design a primer using the Flanking Regions method
1. Open the sequence file you want to analyze.
2. Select the feature of the sequence that defines the flanking regions you are interested on the Map tab or the Features tab. Alternatively, select the region of the sequence that defines the flanking regions you are interested in the Editor tab.
3. Choose Analyze | Primers | Primer Design (Primer3) from the menu.
4. Select Flanking Regions from the design method drop-down list. The values for the left region and the right region are populated based on the sequence selection you made above.
5. The initial values for the flanking regions are zero length so you will need to modify the start point of the left region and the end point of the right region until they encompass the regions you want to choose primers from.
6. Click OK.
Note. There are no limits on the size of the regions that can be used, however, if they are very short then it may not be possible to find suitable primers with the default settings.