We previously described the construction and characterization of Escherichia coli-Francisella tularensis shuttle vectors, derived from the cryptic Francisella plasmid pFNL10, for the genetic manipulation of F. tularensis ssp. tularensis. We now report further characterization of the biology of these shuttle vectors and the development of a new generation of Francisella plasmids. We show that the addition of ORF3 from pFNL10 can convert an unstable shuttle vector into a stable one, and that this is likely due to increased plasmid copy number. We also describe various improvements to the earlier generations of shuttle vectors, such as the addition of a multiple cloning site containing a novel RsrII restriction endonuclease site for directional insertion of Francisella genes, and the inclusion of the F. tularensis blaB promoter for heterologous gene expression.