FIG. 1.

Design of the luciferase-β-galactosidase double-reporter system. (A) pLew20 is a construct containing a wild-type procyclin promoter, a UR (blue), and a 5′ UTR (red) preceding the luciferase ORF (LUC, green). Lowercase letters indicate a small ORF within the 5′ UTR that was carried over from the plasmid from which LUC was excised. pNS11 is a derivative of pLew20 in which the adventitious AUG was deleted from the 5′ UTR and a lacZ reporter gene was added downstream of LUC. In pNS10/54, all four cryptic AG sites were changed to AA or AC (bold) and a HindIII site was introduced at the remaining AG splice site to facilitate the insertion of different URs. pNS10 lacks the UR. (B) Organization of the pNS10/54 reporter plasmid, indicating key components and restriction sites. (C) Luciferase activity in the basic reporter plasmids. All trans-splicing efficiency values shown are measurements of relative luciferase light units normalized to β-galactosidase activity and then to the pNS11 positive control, which was set to 1,000 in this and all subsequent experiments.