Difference between revisions of "Part:BBa K782026"
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* TALA and TALB labels represents TAL effector 1257 and 1297 from zebrafish experiments (Sander et al., 2011). | * TALA and TALB labels represents TAL effector 1257 and 1297 from zebrafish experiments (Sander et al., 2011). | ||
| + | * DNA binding sites for individual TAL effectors are indicated with square brackets [ ]. | ||
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==Introduction== | ==Introduction== | ||
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Our construct contains seven specific binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004 TALA] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB] upstream of minimal promoter. Downstream of minimal 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). | Our construct contains seven specific binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004 TALA] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782006 TALB] upstream of minimal promoter. Downstream of minimal 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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782065 TALA:VP16] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782013 TALB:VP16] on binding sites, an activation of reporter protein mCitrine occurs. | After binding of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782065 TALA:VP16] or [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782013 TALB:VP16] on binding sites, an activation of reporter protein mCitrine occurs. | ||
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| + | Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT | ||
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| + | Single binding site sequence for TALB: TCTTCCGTTTCCACATCT | ||
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* mCitrine was provided from host lab. | * mCitrine was provided from host lab. | ||
| − | * Binding sites for TAL effectors were ordered from | + | * Binding sites for TAL effectors were ordered from GeneArt. |
==References== | ==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, | + | 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. | Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53. | ||
Latest revision as of 21:43, 26 September 2012
7x[TALA+TALB] operator_minimal promoter_mCitrine
- TALA and TALB labels represents TAL effector 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 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 seven specific binding sites for TALA and TALB upstream of minimal promoter. Downstream of minimal 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 TALA:VP16 or TALB:VP16 on binding sites, an activation of reporter protein mCitrine occurs.
Single binding site sequence for TALA: TTTACTGCTGCTCCCGCT
Single binding site sequence for TALB: TCTTCCGTTTCCACATCT
-pMIN-mCit.png){kind=small}
Figure 1: Shematic representation of our construct.
- mCitrine was provided from host lab.
- Binding sites for TAL effectors were ordered from GeneArt.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 488
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 192
Illegal AgeI site found at 55
Illegal AgeI site found at 395 - 1000COMPATIBLE WITH RFC[1000]