Part:BBa_K782016
10x[TALA+TALB] operator_CMV promoter_mCitrine
Introduction
Transcription activation like (TAL) effectors are proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.
Our construct contain ten specific binding sites for TALA and TALB upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm. (Figure 1). After binding of TALA:KRAB or TALB:KRAB con binding sites, a repression of reporter protein mCitrine occurs.
Figure 1. Shematic representation of twelve specific binding site for TALD:KRAB upstream of CMV promoter and reporter protein mCitrine.
Characterization
- mCitrine was provided from 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.
Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 730
Illegal XhoI site found at 1360 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 149
Illegal NgoMIV site found at 514
Illegal AgeI site found at 12
Illegal AgeI site found at 352
Illegal AgeI site found at 377
Illegal AgeI site found at 717 - 1000COMPATIBLE WITH RFC[1000]
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