Part:BBa_K782036

10x[TALA+TALB] operator_CMV promoter_TALD:NLS:KRAB

• TALB, TALA and TALD labels represents TAL effectors 1297 and 1257 and 1295 respectively from zebrafish experiments (Sander et al., 2011).
• DNA binding sites for individual TAL effectors are indicated with square brackets [ ].

Introduction

TAL effectors (TALEs) are bacterial plant pathogen transcription factors, that bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. A tandem TALE repeat contains 33 to 35 amino acids, where the 12th and 13th amino acid, called a “repeat variable diresidue” (RVD), are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).

This part contains 10 alterations of TALA and TALB binding sites (10×[TALA+TALB]), upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter there is a TAL repressor TALD:NLS:KRAB. This way we have a TAL effector under the control of other TAL effectors, which allows us to create more complicated genetic circuits with more than one level of regulation.

Figure 1: Schematic representation of the construct.

References

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.

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.

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 924
  Illegal BamHI site found at 1556
  Illegal BamHI site found at 3924
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 343
  Illegal NgoMIV site found at 708
  Illegal AgeI site found at 206
  Illegal AgeI site found at 546
  Illegal AgeI site found at 571
  Illegal AgeI site found at 911
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4290
  Illegal SapI.rc site found at 4254