You can specify a region within which the amplified product should lie. Simply choose the size of product you want and Primer3 will design primers to amplify products of that size, from anywhere in that region.
To design a primer using the Region to Scan method
1. Open the sequence file you want to analyze.
2. Select a feature of the sequence in the Map tab or the Features tab. Alternatively, select a region of the sequence in the Editor tab.
3. Choose Analyze | Primers | Primer Design (Primer3) from the menu. Note. When the Primer Design (Primer3) dialog box opens it is populated with information about the feature or region you selected in the active sequence window. You can populate the dialog box with information about an alternative feature in the active sequence using the feature selector drop-down menu, or you can specify an alternative region by providing residue numbers manually.
4. Select Region to Scan from the design method drop-down list.
5. Click OK.
By default this method will design primers to produce products between 100 and 300 bases in length. However, you can change the minimum and maximum Product size values to design primers to produce shorter or longer products.