First I needed to take the sequences of the primers I had and find where they existed in the Transcriptome I'm using.
Under primer parameters I put in the forward and reverse primer sequences. Then under Primer Pair Specificity Checking Parameters I changed the Database type to custom and uploaded the Oly Transcriptome V3. Then for added effect I changed the organism to the Ostrea taxa. I don't think this really did much but it didn't detract either. Then I hit get primers and waited for the results.
You can see what the input looks like above. Below is the output for the primers.
This out put tells me the reference name of the template from the Transcriptome on the left, the size of the primer product, and where the primer lays down on the sequence. Under each primer sequence there is a line of ...................... these indicate that the complimentary strand is a 100% match. If any of these dots are replaced with a letter it tells you whats different and that its not a 100% match. You can also see that the top 3 template matches have reference names that are very similar. This is because they are overlapping reads of the same region. If the names were different then we could assume it was a different region.
Once you've got the reference name, then you can follow the same method I used to develop the other flanking primers here.