FIG.2.

pHK77 construction. pHK77 includes the origin of replication of ColE1 to allow replication in E. coli, oriT for conjugational transfer from E. coli to Listeria, and the replication system of pE194ts for temperature-sensitive replication in gram-positive bacteria. There are three selection markers: bla for ampicillin resistance, cat for chloramphenicol resistance, and aadD for neomycin (kanamycin) resistance. Important restriction sites are shown, and unique sites are in bold: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NotI; Nc, NcoI; Nh, NheI; Ns, NsiI; P, PstI; S, SalI; Sa, SacI; Sb, SbfI; Sc, ScaI; Sm, SmaI; Sp, SphI; and X, XbaI.