Reporter

Part:BBa_K782018

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-20)
Revision as of 09:10, 26 September 2012 by UrosZupancic (Talk | contribs)

10x[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 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 TALB:KRAB on binding sites, a repression of reporter protein mCitrine occurs.



10B.png

Figure 1. Schematic representation of the construct.

Characterization

Results: Specific TAL binding sites were further characterized.

Svn 12 10xB-CMV-mCit.png

Figure 2. Repression of TALB:KRAB.


  • 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 720
    Illegal XhoI site found at 1350
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 147
    Illegal NgoMIV site found at 507
    Illegal AgeI site found at 12
    Illegal AgeI site found at 347
    Illegal AgeI site found at 372
    Illegal AgeI site found at 707
  • 1000
    COMPATIBLE WITH RFC[1000]


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