FIG. 1.

Construction of IVET plasmids pRIV16 and pRIV16C. (A) Restriction map of a promoterless transcriptional fusion between pyrBC′ and lacZ. (B) pRIV16 and pRIV16C carry pyrBC′-lacZ in the reverse orientation. Promoterless pyrBC′-lacZ digested with BamHI and partially digested with HindIII was cloned into pRK415 to generate pRIV16 and was cloned into a XbaI site as a blunt-end ligation to generate pRIV16C. The restriction enzyme sites in the pyrBC′-lacZ genes are not shown for pRIV16 and pRIV16C. The SphI (Sp) and PstI (P) sites in the upstream region of pyrBC′ were derived from pUC119 in the subcloning process. The polylinker sites between pyrBC′ and lacZ were originally from pHSK728. The boldface BamHI (B), XbaI (X), and HindIII (H) sites are cloning sites in pRK415. The boldface BamHI (B) and XbaI (X) sites in pRIV16C were retained after blunt-end ligation. The enzyme sites located outside the boldface letters were derived from the polylinker of pRK415. Abbreviations: B, BamHI; C, ClaI; E, EcoRI; Ev, EcoRV; H, HindIII; Hc, HincII; K, KpnI; P, PstI; S, SalI; Sc, SacI; Sm, SmaI; Sp, SphI; X, XbaI; Xh, XhoI.