Part:BBa_K782020

10x[TALA] operator_CMV promoter_fLuciferase

TALA label represents TAL effector 1257 from zebrafish experiments (Sander et al., 2011)

Introduction

Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011)

We designed our construct with 10 binding sites for TALA, upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.

Figure1:Schematic representation of our construct

Characterisation

HEK293T cells were transfected with the 10x[TALA] operator_CMV promoter_fLuciferase reporter and TALA:NLS:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with either TAL repressor.

Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALA:KRAB.

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 914
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 341
  Illegal NgoMIV site found at 701
  Illegal NgoMIV site found at 1626
  Illegal NgoMIV site found at 2970
  Illegal NgoMIV site found at 2991
  Illegal NgoMIV site found at 3306
  Illegal AgeI site found at 206
  Illegal AgeI site found at 541
  Illegal AgeI site found at 566
  Illegal AgeI site found at 901
  Illegal AgeI site found at 2694
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2876