pNLCoI3[luc2-P2A-NlucP/CMV/Hygro] Vector

Two Reporter Genes, One Transcript

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 sequentially detects firefly and NanoLuc® Luciferase in activity in the same sample with a convenient “add-read-add-read” homogeneous format. Both NanoDLR™ reagents provide stable glow-type luminescence signals with half-lives of approximately two hours for automated batch processing of samples and amenable to assays or high-throughput 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.

pNLCoI1[luc2-P2A-NlucP/Hygro] Vector GenBank® Accession Number KM359771 and vector sequence text file.

pNLCoI2[luc2-P2A-NlucP/minP/Hygro] Vector GenBank® Accession Number KM359772 and vector sequence text file.

pNLCoI3[luc2-P2A-NlucP/CMV/Hygro] Vector GenBank® Accession Number KM359773 and vector sequence text file. 

pNLCol4[luc2-P2A-NlucP/PGK/Hygro] Vector  GenBank® Accession Number KM376434 and vector sequence text file.