Part:BBa_K782014

12x[TALD] operator_CMV promoter_mCitrine

• TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).

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 contains twelve specific binding sites for TALD 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 TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs.

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC

Figure 1. Shematic representation of twelve specific binding site for TALD:KRAB upstream of CMV promoter and reporter protein mCitrine.

Characterization

Results: Specific TAL binding sites were further characterized with other reporter constructs.

• mCitrine was provided from host lab.
• Binding sites for TAL effectors were ordered from GeneArt.

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 465
  Illegal XhoI site found at 1095
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 214
  Illegal AgeI site found at 446
  
• 1000
  COMPATIBLE WITH RFC[1000]