TCCTCAAATTACATACGCCTACCTTGTTAATACTTCCGATTCCTTTTC CTCTTCTCTCAGAACCCATTTTTAGAGGTCAGAGTTACAGACACACCGAAACGGTCCCGC AGAGATTTTGGCCTTGACTGTGATGAGCACTCAACGGAATCCCGATGTTGTCGCTACCCG CTGACAGTGGATTTCGAAGCTTTTGGATGGGACTGGATTATAGCACCTAAAAGATACAAA GCCAATTACTGCTCCGGAGAATGCGAATTTGTGTTTCTACAGAAATACCCGCACACTCAC CTGGTACACCAAGCAAATCCCAGAGGCTCAGCAGGCCCTTGCTGCACACCCACCAAGATG TCCCCTATAAACATGCTGTATTTCAATGGAAAAGAACAAATAATATATGGAAAGATACCA GCCATGGTTGTAGATCGTTGCGGGTGCTCATGAGGCTGTCGTGAGATCCACCATTCGATA AATTGTGGAAGCCACCAAAAAAAAAAGCTATATCCCCTCATCCATCTTTGAAACTGTGAA ATTACGTACGCTAGGCATT
First, I put this into Primer3 with '60,427' in the Targets field. This worked out great and I got a couple of primers which I then put into BLAT to ensure they didn't occur elsewhere in the Chicken genome.
Then I discovered Primer-Blast which is uses Primer3 and Blast together, which I figured would make the whole thing a lot easier. I put in my sequence and the limits 1 to 60 for forward and 487 to 547 for reverse primers and specified the organism (Gallus gallus) whose genome ought be checked against.
In return, I received the error:
No primers were found...see explanation below: Primer3 info: Left primer: considered 451, low tm 451, ok 0. Right primer: considered 380, too many Ns 6 (This could be due to low complexity and/or repeat filtering. Try search with filtering off), low tm 374, ok 0. Primer pairs: considered 0, ok 0
The Primer-Blast interface doesn't have any option for repeat filtering. Could anyone offer some insight?