Patent ID: 8728760

Claim:
An expression vector comprising a nucleotide sequence encoding: (a) a restricted-copy-number origin of replication cassette comprising (i) a nucleotide sequence encoding an origin of replication that limits the expression vector to an average plasmid copy number of about 2 to 75 copies per cell, (ii) a first unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the origin of replication, and (iii) a second unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the origin of replication; (b) at least one post-segregational killing cassette comprising (i) a nucleotide sequence encoding at least one post-segregational killing locus, (ii) a third unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the at least one post-segregational killing locus, and (iii) a fourth unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the at least one post-segregational killing locus; (c) at least one partitioning cassette comprising (i) a nucleotide sequence encoding at least one partitioning function, (ii) a fifth unique restriction enzyme cleavage site 5′ of the nucleotide sequence encoding the at least one partitioning function, and (iii) a sixth unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the at least one partitioning function; and (d) at least one selectable marker cassette comprising (i) a nucleotide sequence encoding at least one selectable marker, wherein said selectable marker is the E. coli polypeptide mchI having the amino acid sequence set forth in SEQ ID NO:30 (ii) a seventh unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the at least one selectable marker, and (iii) an eighth unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the at least one selectable marker.