1 atgcaggcca aaaaacgcta tttcatcctg ctctcagctg gctcttctct cgcccttttg
61 ttttattttg gaggcgtgca gtttagggca tcgaggagcc acagccggag agaagagcac
121 agtggtcgga atggcttgca ccagcccagt ccggatcatt tctggccccg cttcccggag
181 cctctgcgcc ccttctttcc ttgggatcaa ttggaaaacg aggattccag cgtgcacatt
241 tccccccggc agaagcgaga cgccaactcg agcatctaca aaggcaagaa gtgccgcatg
301 gagtcctgct tcgatttcac cctttgcaag aaaaacggct tcaaagtcta cgtgtacccg
361 cagcagaaag gggagaaaat cgccgaaagt taccaaaaca ttctagcggc catcgagggc
421 tccaggttct acacctcgga ccccagccag gcgtgcctct ttgtcttgag tctggatact
481 ttagacagag accagttatc acctcagtat gtgcacaatt tgagatccaa agtgcagagt
541 ctccacttgt ggaacaatgg taggaatcat ttaattttta atttatattc tggcacttgg
601 cctgactaca ctgaggacgt ggggtttgac atcggccagg cgatgctggc caaagccacg
661 atcagtactg aaaacttccg accaaacttt gatgtttcta ttcccctctt ttctaaggat
721 catcccagga caggagggga gagggggttt ttgaaattta acaccatccc tcctctcagg
781 aagtacatgc tggtattcaa ggggaagcgg tacctgacag ggatagggtc agacaccagg
841 aatgccttat atcacgtcca taacggggag gacgtcttgc tcctcaccac ctgcaagcat
901 ggcaaagact ggcaaaagca caaggattct cgctgtgaca gagacaacac cgagtatgag
961 aa

Primer3 Primer Design Protocol

http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

1. Enter exon sequence.

2. Restrict primer temperature to 59-61ºC.

3. Restrict primer size to 19-21 base pairs.

4. Change the following parameters:

a. Product Size Ranges – 65-75 base pairs
b. Max Tm Difference – 1C
c. Max Self Complementarity - 6
d. Max 3’ Complementarity - 5
e. Max Poly-X - 3
f. %GC Content – 20-80%
g. Number to return - 5 (but can be changed as needed)

5. Program defaults used for all other options.

Validated primers: (07/21/06)

Left primer: TGGAGTCCTGCTTCGATTTC
Right primer: CTTTCTGCTGCGGGTACAC