In vivo and in vitro recombination techniques were used to construct a new cloning vector, pHY300PLK (4.7 kb) from the shuttle vector pHY460 (7 kb). The newly derived shuttle vector can replicate and express the tetracycline resistance gene (TcR) in both Escherichia coli and Bacillus subtilis. pHY300PLK contains the TcR gene, the ampicillin resistance gene (ApR), two replication origins for EK coli and B. subtilis and a polylinker derived from ~AN7. The unique cloning sites are BalI,

A small plasmid pHY163PLK (2519 bp) was constructed from the shuttle vector pHY300PLK. pHY163PLK contains the replication origin of pACYC-177, the tetracycline resistance gene (tetal) of pAMal derived from Streptococcus faecalis and eight continuous unique cloning sites of a polylinker region (EcoRI, SmaI, BamHI, SalI, PstI, BglII, XbaI and HindIII), pHY163PLK, a copy-number mutant, has one base substitution in RNA I region compared with that of p15A which is the ancestor of ori-pHY163PLK.The tetal, which codes 458 amino acids (1374 bp), is highly homologous to the TcR genes of other Gram-positive bacteria but completely different from the TcR gene encoded by pBR322 in Gram-negative bacteria. The regulatory region of tetal (constitutive type) has an insertion of seven base pairs in the leader peptide region of a related inducible TcR