Fig 2.

Cassettes used in vector construction. Added restriction sites are indicated by a plus sign before the restriction site, whereas a slash through the restriction site indicates that the restriction site was removed by a silent point mutation. The two ClaI sites used for the deletion in pRLYT8 are indicated. (A) The Pspac^C-pyrFE cassette, with pyrF and pyrE genes from B. subtilis. The C-to-T point mutation used to avoid instability in E. coli is indicated. The −10 box, the transcription start site, and the start codon are indicated in bold type. The 6-bp repeats are underlined, and the asterisk indicates where we introduced the C-to-T point mutation. The three HindIII sites and a BglII site were removed from the pyrFE genes by site-directed mutagenesis to maintain the uniqueness of the corresponding sites. (B) Antibiotic resistance cassettes used for construction of the pRLYx1 vectors. An NcoI site in CAT194 and a SacI site in ermC were removed by site-directed mutagenesis via silent mutations. (C) Origins of replication used for construction of the pRLYx2, pRLYx8, and pRLYx9 vectors. ORI₁₈₁ (ORI) is indicated, and broken lines indicate the section of the full-length pT181ts origin of replication, which comprises the ORI₁₈₁, used for the pRLYx2 vectors. The XbaI site was mutated in the pT181ts origin.