I work with reverse genetic systems for RNA viruses. It is very difficult to clone viral genes, since our viruses harbour GC rich sequences. Complete cDNAs are larger than 16 kb and are usually cloned in pBR322, because a Rob gene is needed for stability. These plasmids are exclusively proliferated in the old E. coli strain HB101 which accepts such ugly bugs, but the bacteria grow very tiny colonies. At the end of the day most of the plasmids still grow very poor
, DNA is not easy to preparate
and mutations are frequently generated
. So my question:
Does anybody know more stable vectors for such large inserts?