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 bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD) are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed twelve specific binding sites for TALD:KRAB upstream of CMV promoter (Figure 1). After binding of TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs. mCitrine is yellow fluorescent protein.

Single binding sequence for TALD:KRAB is: TCGTCCAATAGCTTCTC

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

• 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 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]