Extreme miniaturization in plasmid design: generation of the 903 bp cloning vector pICOt2

Inspired by the size optimizations in the recently published pUdO series of plasmids, we set out to break our previous world record (pICOz, 1185 bp) in designing the smallest possible self-replicating high copy cloning vector. By replacing the zeocin resistance marker with trimethoprim resistance, deleting the parts upstream of -35 in the ampicilin resistance promoter and replacing the extended multiple cloning site with a single SpeI site, we managed to reduce the plasmid size to 903 bp, almost a 24% reduction in size compared to our previous accomplishments.