The pNLCoI Vectors comprise a second-generation coincidence reporter vector system that allow expression of both firefly luciferase (luc2) and NanoLuc Luciferase fused to a PEST destabilization domain (NlucP) from the same mRNA transcript.

• Vectors That Express Both Firefly and NanoLuc(R) Luciferases from the Same Transcript
• Firefly and NanoLuc(R) luciferase have dissimilar compound interference profiles for better identifying more false-positives versus individually
• Using two different transcriptional reporters reduces false hit rates and increases the identification of true biological hits
• luc2 and NlucP provide a bright reporter combination compatible with low-copy-number and plate scale-up, and provide greater signal-to-background ratios

Luciferase-based reporter-gene assays remain a useful and powerful method of high-throughput compound screening. However, false hits that result from direct interaction of compounds with the luciferase reporter can result in unnecessary follow-up efforts. The pNLCoI Vectors comprise a second-generation coincidence reporter vector system that allow expression of both firefly luciferase (luc2) and NanoLuc Luciferase fused to a PEST destabilization domain (NlucP) from the same mRNA transcript. The stoichiometric expression of both luciferases is achieved by use of the P2A sequence from porcine teschovirus-1, which promotes a ribosomal skip and expression of the two unfused enzymes with distinct compound interaction profiles. When used in high-throughput compound screening, false hits caused by direct interaction with one or the other luciferases can be distinguished from true hits that show a similar response for both, reducing workload associated with follow-up screens. The pNLCoI Vectors are designed for use with the Nano-Glo Dual-Luciferase Reporter (NanoDLR) Assay System, which allows sequential detection of firefly and NanoLuc Luciferase in activity in the same sample. Both reagents provide stable glow-type luminescence signals with half-lives of approximately two hours allowing batch processing of samples and amenable to assays or screens in 96-, 384- or 1,536-well plate formats. Potent inhibition of firefly luciferase coupled with the high-intensity luminescence of NanoLuc luciferase maximizes sensitivity for detection of both reporters.