Part:BBa_K4218004:Design
The dual luciferase reporter system
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2459
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 3661
Design Notes
To measure the alteration of RNA splicing, we engineered one dual luciferase reporter system where two luciferase genes were fused with GTGAGT-recessive exon MAP3K7-CCACAG minigenes . Upon transfection of the dual luciferase reporters into mammalian cells, the pre-mRNA of this dual reporter would be processed in either of two ways. During normal splicing in cells, the internal stop codon would be removed, then the upstream reporter gene (Firefly luciferase, Fluc) and the downstream reporter gene (Renilla luciferase, Rluc) will be placed in the same reading frame to generate a fusion protein Fluc-Rluc. In the case that splicing in inhibited by splicing inhibitors, the stop codon in the recessive exon would lead to the translation termination of the mRNA, thus producing the Fluc protein alone. Therefore, the Fluc gene is expressed regardless of whether splicing occurred, whereas the downstream Rluc gene can only be expressed after splicing.
Source
Uses a positively regulated promoter(BBa_K337013),our basic part BBa_K4218013，BBa_K4218011, BBa_K4218014, anotherpositively regulated promoter(BBa_K3064008),Firefly Luciferase (BBa_J96034)