Part:BBa_K782000

7x[NicTAL]+7x[TALD] operator_CMV promoter_mCitrine

Introduction

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

Figure 1. Shematic representation of seven consecutive specific binding sites for NicTAL and TALD upstream of CMV promoter and reporter protein mCitrine.

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 (link). We designed seven consecutive specific binding sites for NicTAL and TALD upstream of CMV promoter, that ensure inducible expression of reporter protein mCitrine. mCitrine is yellow fluorescent protein.

Single binding sequence for NicTAL is: ACTCTATCAATGATAGAGT

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC

• 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 478
  Illegal XhoI site found at 1108
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 35
  Illegal AgeI site found at 443
  
• 1000
  COMPATIBLE WITH RFC[1000]