Difference between revisions of "Part:BBa K782003"
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[[Image:11x.png]]
 
[[Image:11x.png]]
  
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Figure 1. Shematic representation of two consecutive specific binding sites for NicTAL and TALD  
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Figure 1. Shematic representation of specific binding site for NicTAL and TALD  
 
upstream of CMV promoter and reporter protein mCitrine.  
 
upstream of CMV promoter and reporter protein mCitrine.  
  
  
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Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD)  are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).  All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed two consecutive specific binding sites for NicTAL and TALD upstream of CMV promoter, that ensure inducible expression of reporter protein mCitrine. mCitrine is yellow fluorescent protein.  
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Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD)  are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011).  All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed specific binding site for NicTAL and TALD upstream of CMV promoter, that ensure inducible expression of reporter protein mCitrine. mCitrine is yellow fluorescent protein.  
  
 
Single binding sequence for NicTAL is:  ACTCTATCAATGATAGAGT   
 
Single binding sequence for NicTAL is:  ACTCTATCAATGATAGAGT   

Revision as of 13:47, 25 September 2012

1x[NicTAL]+1x[TALD] operator_CMV promoter_mCitrine

  • TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).


11x.png

Figure 1. Shematic representation of specific binding site for NicTAL and TALD upstream of CMV promoter and reporter protein mCitrine.


Transcription activation like (TAL) effectors are bacterial plant pathogen transcription factors that bind to DNA by recognizing a specific DNA sequence in which each base pair binds a single tandem repeat in in the TAL DNA-binding domain. A tandem TAL repeat contains 33 to 35 amino acids, where the 12th and the 13th amino acid, called a “repeat variable diresidue” (RVD) are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). All TAL repeats have almost identical sequences, differing only in the RVDs. This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences by designing specific binding domains for a selected TAL effector. We designed specific binding site for NicTAL and TALD upstream of CMV promoter, that ensure inducible expression of reporter protein mCitrine. mCitrine is yellow fluorescent protein.

Single binding sequence for NicTAL is: ACTCTATCAATGATAGAGT

Single binding sequence for TALD is: TCGTCCAATAGCTTCTC


  • mCitrine was provided from host lab.
  • Binding sites for TAL effectors were ordered from IDT.


References

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

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
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 70
    Illegal XhoI site found at 700
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 35
  • 1000
    COMPATIBLE WITH RFC[1000]