Difference between revisions of "Part:BBa K782003"
(→Introduction) |
|||
Line 1: | Line 1: | ||
__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K782003 short</partinfo> | <partinfo>BBa_K782003 short</partinfo> | ||
+ | |||
* TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011). | * TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011). | ||
+ | * DNA binding sites for individual TAL effectors are indicated with square brackets [ ] | ||
+ | |||
==Introduction== | ==Introduction== | ||
Line 26: | Line 29: | ||
* mCitrine was provided from host lab. | * mCitrine was provided from host lab. | ||
* Binding sites for TAL effectors were ordered from GeneArt. | * Binding sites for TAL effectors were ordered from GeneArt. | ||
+ | |||
==References== | ==References== |
Revision as of 12:39, 26 September 2012
1x[NicTAL]+1x[TALD] operator_CMV promoter_mCitrine
- TALD label represents TAL effector 1295 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 proteins able to specifically bind desired DNA sequence. The central domain of the protein is constructed from variable number of tandem repeats differing only in two amino acids. The 12th and the 13th amino acid are called a “repeat variable diresidue” (RVD) and are responsible for specific interactions with the corresponding base pair (Scholze and Boch, 2011). This modularity of TAL effector binding domains therefore makes them a perfect tool to target specific DNA sequences.
Our construct contains specific binding site for NicTAL12 and TALD upstream of CMV promoter. Downstream of CMV promoter we cloned yellow fluorescent protein mCitrine an easy detectable monomer with excitation maximum at 516 nm and emission maximum at 529 nm. (Figure 1). After binding of NicTAL12:KRAB or TALD:KRAB on binding sites, a repression of reporter protein mCitrine occurs.
Single binding sequence for NicTAL12 is: TCTATCAATGATAGA
Single binding sequence for TALD is: TCGTCCAATAGCTTCTC
Figure 1. Shematic representation of specific binding site for NicTAL12:KRAB and TALD:KRAB upstream of CMV promoter and reporter protein mCitrine.
- mCitrine was provided from host lab.
- Binding sites for TAL effectors were ordered from GeneArt.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 70
Illegal XhoI site found at 700 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 35
- 1000COMPATIBLE WITH RFC[1000]