Part:BBa_K782016

10x[TALA+TALB] operator_CMV promoter_mCitrine

• TALA and TALB labels represents TAL effector 1257 and 1297 respectively from zebrafish experiments (Sander et al., 2011).
• DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

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 10 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 on binding sites, a repression of reporter protein mCitrine occurs.

Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT

Single binding site sequence for TALB: TCTTCCGTTTCCACATCT

Figure 1. Shematic representation of ten alternating specific binding site for TALA and TALB upstream of CMV promoter and reporter protein mCitrine.

Characterization

• 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 730
  Illegal XhoI site found at 1360
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE WITH RFC[1000]