Recombinant vector with primer sites and alternate insert orientations. For sequencing, the products generated by TVL-PCR must be cloned into the TOPO vector. Two possible insert orientations are possible, as shown. M13F or M13R primers must be used for sequencing because they provide unique priming sites in the recombinant plasmid. The T3 or T7 primers are unsuitable for sequencing because they are represented in both the vector and the insert.
Orcheski and Davis BMC Research Notes 2010 3:61 doi:10.1186/1756-0500-3-61