I have been trying to design PCR primers that span a splice junction using the NCBI Primer design tool for a gene (NW_005179589.1). It seems like said gene has 4 exons. When I go to pick primers, I select the "Primer must span an exon-exon junction" option, but even when I have everything else on default settings, I get the error message:
"No primers were found...see explanation below: Exon/exon junction cannot be found for submitted PCR template. Try search again without exon/intron requirements."
Am I missing something here? There are several introns in the gene so I'm confused as to why I'm getting this message.
Any help would be appreciated.