Restriction digests of the clone give the following sizes (kb): HindIII-- 2.7; XbaI-- 2.7; SacI/XhoI-- 2.4, 0.3. The mini-exon is flanked by consensus 3' and 5' splice sites. The exon contains open reading frames encoding 43 amino acid peptides. There are no stop codons in any of the three reading frames, and each reading frame contains an epitope recognized by the same monoclonal antibody. The mini-exon is cloned into a plasmid with the tetracycline-resistance marker, which facilitates further subcloning. pBR327-myc2 is a vector that contains a mini-exon that can be inserted into introns, permitting the detection of gene products using the same monoclonal antibody (9E10) regardless of intron class. The vector was constructed by inserting the 170-nucleotide sequence, synthesized and then PCR amplified, into pBluescript-KS(+) and then excised using KpnI and SacI and ligated into pUC1813. The mini-exon was then excised as a blunt SmaI fragment and ligated into pBR327, which had been cut with EcoRI and BsaI, the ends having been filled in using Klenow. |
