Three origins of replication are active in vivo in the R plasmid RSF1040.

Replicating DNA molecules of RSF1040, a deletion derivative of the conjugative R plasmid R6K, are cleaved at a single site by the Eco RI restriction endonuclease. Microscopic analysis of Eco RI-cleaved RSF1040 replicative intermediates synthesized in vivo indicates that initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma. The relative frequencies of initiations at these three origins are different from those found in vitro.