Part:BBa_K5490017
Ca-Dependent Synthetic NF-AT
Is a synthetic NFAT transcription factor, after an increase in calcium, will enter the nucleus and bind to a specific minimal promoter. It uses the TALE system for binding to DNA and VP16 as the activation domain. Under homeostatic conditions, it is anchored in the plasma membrane via the KRφ peptide. It has both Myc and FLAG tags for immunohistochemical analysis and Western blotting
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 723
Illegal EcoRI site found at 766
Illegal EcoRI site found at 1926
Illegal SpeI site found at 741
Illegal PstI site found at 1744
Illegal PstI site found at 1998 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 723
Illegal EcoRI site found at 766
Illegal EcoRI site found at 1926
Illegal NheI site found at 1315
Illegal SpeI site found at 741
Illegal PstI site found at 1744
Illegal PstI site found at 1998
Illegal NotI site found at 715 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 723
Illegal EcoRI site found at 766
Illegal EcoRI site found at 1926
Illegal BamHI site found at 1793
Illegal BamHI site found at 4543
Illegal XhoI site found at 2032 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 723
Illegal EcoRI site found at 766
Illegal EcoRI site found at 1926
Illegal SpeI site found at 741
Illegal PstI site found at 1744
Illegal PstI site found at 1998 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 723
Illegal EcoRI site found at 766
Illegal EcoRI site found at 1926
Illegal SpeI site found at 741
Illegal PstI site found at 1744
Illegal PstI site found at 1998
Illegal NgoMIV site found at 1198
Illegal NgoMIV site found at 1218
Illegal NgoMIV site found at 1234
Illegal NgoMIV site found at 1699
Illegal NgoMIV site found at 1983 - 1000COMPATIBLE WITH RFC[1000]
NFAT Transcription Factor cn Domain:
In its natural state, NFAT contains two Nuclear Localization Signals (NLS) that enable nuclear translocation. However, in this engineered version, one NLS has been removed, leaving only a single NLS to reduce background transcription. The NFAT domain is activated through phosphorylation by calcineurin in response to calcium signaling. This domain is critical for calcium-dependent activation in cellular processes, as the increase in cytosolic calcium activates calmodulin, which, in turn, activates calcineurin.
TALE Binding Domain and Calcineurin Fusion:
The TALE domain, which recognizes specific DNA sequences, is fused to the calcineurin domain via a GS10 flexible linker. The TALE sequence has high affinity for 10 TALE binding sites, which can be placed at various positions within the DNA, such as upstream of the TATA box to activate transcription of target genes.
VP16 Activation Domain and KRφ Peptide:
Transcription activation is achieved through the VP16 activation domain, which recruits the necessary cellular machinery, such as RNA polymerase II, to the promoter region. To prevent background transcription, a KRφ peptide is fused to the C-terminus of the protein, which anchors the protein to the inner plasma membrane. Upon elevated calcium levels, the protein is released from the membrane and translocates to the nucleus via the remaining NLS, initiating transcription. After calcium levels normalize, the protein is dephosphorylated and returns to the cytosol, where it re-anchors to the membrane, resetting for further activation.
The chimeric protein is also tagged at the N-terminus with Myc and FLAG tags to facilitate detection and purification using techniques like Western blotting, immunohistochemistry, and affinity purification. Overexpression of this chimeric protein is recommended to achieve robust promoter activation and significant transcriptional output
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