Reporter

Part:BBa_K782033

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-20)

10x[TALA] operator_minimal promoter_fLuciferase

  • TALA and TALB labels represents TAL effectors 1257 and 1297 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 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 minimal promoter (like here). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.

10×A pMIN fLuc shema1.png

Figure 1:Schematic representation of our construct.


Characterisation

10x[TALA] operator_minimal promoter was characterised with mCitrine reporter.

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 XhoI site found at 1004
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 393
    Illegal NgoMIV site found at 753
    Illegal NgoMIV site found at 1102
    Illegal NgoMIV site found at 2446
    Illegal NgoMIV site found at 2467
    Illegal NgoMIV site found at 2782
    Illegal NgoMIV site found at 2890
    Illegal AgeI site found at 258
    Illegal AgeI site found at 593
    Illegal AgeI site found at 618
    Illegal AgeI site found at 953
    Illegal AgeI site found at 2170
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2352


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