Part:BBa_K782023
12x[NicTAL] operator_CMV promoter_fLuciferase
- DNA binding sites for individual TAL effectors are indicated with square brackets [ ]
Introduction
Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011)
We designed our construct with 12 binding sites for NicTAL (as described here), upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.
Figure1:Schematic representation of our construct
Characterisation
HEK293T cells were transfected with the 12x[NicTAL] operator_CMV promoter_fLuciferase reporter and NicTAL:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).
Figure 2:Schematic representation of repression experiments. A: in the absence of a NicTAL:KRAB, fLuciferase is constituitively expressed. B: when a NicTAL:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.
Figure 3:Testing repression of reporter luciferase gene. Light-blue column is showing constantly expressed reporter (fLuc), dark-blue column is showing repression of fLuc reporter with NicTAL:KRAB.
- fLuciferase was contributed by host lab
- Binding sites for TAL effectors were ordered from IDT.
References
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 658
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1370
Illegal NgoMIV site found at 2714
Illegal NgoMIV site found at 2735
Illegal NgoMIV site found at 3050
Illegal AgeI site found at 210
Illegal AgeI site found at 442
Illegal AgeI site found at 2438 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2620
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