We report the construction of a series of replicating shuttle vectors that 
consist of a low-copy-number cloning vector for Escherichia coli and functional 
components of the origin of replication (oriC) of the chromosome of the 
hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying 
the minimum replication origin sequence required for autonomous plasmid 
replication in P. furiosus, we discovered that several features of the origin 
predicted by bioinformatic analysis and in vitro binding studies were not 
essential for stable autonomous plasmid replication. A minimum region required 
to promote plasmid DNA replication was identified, and plasmids based on this 
sequence readily transformed P. furiosus. The plasmids replicated autonomously 
and existed in a single copy. In contrast to shuttle vectors based on a plasmid 
from the closely related hyperthermophile Pyrococcus abyssi for use in P. 
furiosus, plasmids based on the P. furiosus chromosomal origin were structurally 
unchanged after transformation and were stable without selection for more than 
100 generations.