Exontrap Cloning Vector pET01

The Exontrap vector pET01 has an intrinsic splicing function that enables selective cloning of exon sequences from large genomic eukaryotic DNA fragments. This new route for the identification of eukaryotic genes does not involve an initial isolation of cellular mRNA, allowing genes that are not transcribed during certain life cycle stages to be identified. This greatly facilitates exon/intron mapping. 

The Exontrap function is based on a shuttle vector containing prokaryotic and eukaryotic genetic elements for replication in both bacteria and cell cultures. The vector contains a 5´ and 3´ exon separated by a 600 bp intron sequence that has a polylinker for cloning. The recombinant vector is transfected into eukaryotic cells (e.g. COS cells) and transcribed. Restriction or sequence analysis can determine if the insert contains an exon in the correct orientation. As the mRNA is processed, the intron sequences originating from the vector, as well as those being introduced, are removed. After total RNA isolation, the mature mRNA is reverse transcribed into cDNA using a specific primer complementary to a sequence of the bordering exon. The cDNA is amplified by PCR using specific primers, which create restriction sites for further subcloning.