The development of pBR322 plasmid cloning vector involved a systematic strategy of modifying natural plasmids to enhance their utility for gene cloning.

Plasmids originally found in nature are modified, shortened, reconstructed and recombined under both in vivo and in vitro conditions to incorporate desirable properties. The precursor plasmid pBR313 was developed first, which replicated in a relaxed fashion giving rise to high copy number. It contained two selectable markers — tetracycline resistance ($tet^{R}$) and ampicillin resistance ($amp^{R}$) derived from other naturally occurring plasmids, and carried a number of unique restriction sites (cloning sites).

However, the size of pBR313 was too large at 9 kb. It was observed that more than half of its DNA was not essential for its role as a vector. Therefore, the first phase of plasmid vector development led to the construction of pBR322, whose size was reduced to 4,361 bp by deleting most of the non-essential sequences from pBR313. The strategy ensured that pBR322 fulfilled key features required for an ideal vector: small size for easy incorporation into host cells, presence of origin of replication (ori) for autonomous replication, unique restriction sites to avoid fragmentation, and selectable markers to screen transformants. The relaxed replication of this high copy number plasmid gives higher yields and increased signal strength during screening of recombinants.

Thus, pBR322 became the most widely used vector for gene cloning.