Part:BBa_K782087

10x[TALA] operator_minimal promoter

• TALA and TALC labels represents TAL effector 1257 and 1259 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 TALA binding sites 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 specificly recognised by TALA. Binding sites are constructed in alternating setup. Besides TALA binding sites construct also contain ten alternating TALC binding sites, which we didn't use, since TALC regulator wasn't working properly.

Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT

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 of TALA.

Results:

• BBa_K782029

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 394
  Illegal NgoMIV site found at 754
  Illegal AgeI site found at 259
  Illegal AgeI site found at 594
  Illegal AgeI site found at 619
  Illegal AgeI site found at 954
  
• 1000
  COMPATIBLE WITH RFC[1000]