Part:BBa_K782086

10x[TALA+TALB] operator_minimal promoter

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

Introduction

TAL effectors (TALEs) are able to specifically bind to the DNA by recognizing base pairs on a DNA molecule. With TAL regulators we are able to control expression of a desired gene. Efficient transcription control can be achieved by binding of multiple TAL regulators upstream of promoter.

Our construct contain ten binding sites for TALA, another ten for TALB and minimal promoter. Minimal promotor is taken from pGL4 vector series (promotor is described [http://www.promega.com/~/media/files/resources/protocols/technical%20manuals/0/pgl4%20luciferase%20reporter%20vectors%20protocol.pdf?la=en here] on the page 18).

Single binding site is combined from 18 base pairs recognised by specific TAL. TALA and TALB binding sites are adjacent to each other making impossible for both of them to bind at once.

Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT

Single binding site sequence for TALB: TCTTCCGTTTCCACATCT

Figure 1: Schematic representation of the construct.

• Binding sites for TAL effectors were ordered from IDT.

Characterization

Construct was later used for constructing different reporter plasmids to show specific binding and competition between TALA and TALB.

Results:

• BBa_K782028
• BBa_K782032

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 396
  Illegal NgoMIV site found at 761
  Illegal AgeI site found at 259
  Illegal AgeI site found at 599
  Illegal AgeI site found at 624
  Illegal AgeI site found at 964
  
• 1000
  COMPATIBLE WITH RFC[1000]