Part:BBa_K782084
10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune
- TALB and TALA labels represents TAL effectors 1297 and 1257 respectively from zebrafish experiments (Sander et al., 2011).
- DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
Introduction
This part combined with 10x[TALB] operator_CMV promoter_TALA:KRAB:NLS_t2a_mCitrine is the key element of the [http://2012.igem.org/Team:Slovenia/TheSwitchMutualRepressorSwitch mutual repressor switch].
The part contains 10 repeats of TALA binding sites upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter, there is the TAL repressor TALB:KRAB linked to the red fluorescent protein mNeptune, by a t2A sequence. The t2A sequence, causes the ribosome to skip the formation of a peptide bond during protein translation, producing TALB:KRAB and mNeptune as separate proteins in equimolar amounts (Garg et.al, 2012). mNeptune functions as a reporter for the expression of TALB:KRAB, it is a red fluorescent protein with an excitation peak at 600 nm and emission peak at 650 nm and brightness of 13.4.
Figure 1: Schematic representation of the construct.
Characterization
HEK293T cells were transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune and visualized under confocal microscope. Cells were shown to fluoresce with mNeptune, showing that mNeptune is expressed.
Figure 2: Cells transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune, fluorescing with mNeptune.
HEK293T cells were transfected with 10×[A]_PCMV_TALB:KRAB_mNeptune, 10×[B]_PCMV_TALA:KRAB_mCitrine, 10×[B]_PCMV_fLuciferase reporter (firefly luciferase) and different amounts of PCMV_TALA:KRAB. TALA:KRAB represses TALB:KRAB causing derepression of fLuciferase, showing that the construct is repressed by TALA:KRAB.
Figure 3: Repression of TALB:KRAB, by TALA:KRAB.
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.
Garg, A., Lohmueller, J. J., Silver, P. A. and Armel, T. Z. (2012) Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 7584-7595.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 720
Illegal BamHI site found at 3773 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 147
Illegal NgoMIV site found at 507
Illegal AgeI site found at 12
Illegal AgeI site found at 347
Illegal AgeI site found at 372
Illegal AgeI site found at 707 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4139
Illegal BsaI.rc site found at 4688
Illegal BsaI.rc site found at 4877
Illegal SapI.rc site found at 4103
Illegal SapI.rc site found at 4259
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