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]
IMPORTANT NOTICE
The NFAT construct was kindly provided by Benčina M, with the goal of studying its translocation to the nucleus after ionophore stimulation. For detection, we used an immunohistochemical approach targeting the Myc-tag epitope, which was fused to the N-terminus of the NFAT construct. However, the antibody against the Myc-tag epitope generated significant background noise. While it had some affinity for its target, it also bound nonspecifically to other molecules, leading to an inconclusive image under the microscope. Despite this issue, we confirmed the expression of the synthetic protein via Western blot, which motivated us to explore alternatives. Specifically, we decided to add a Flag tag in front of the Myc-tag epitope, leading us to try two different cloning strategies to achieve this.
First, we attempted to fuse the Flag tag using subcloning. In this approach, we extracted the insert and blunt-ended one side of both the vector and the insert while keeping the other side sticky to maintain directionality during the ligation step. Unfortunately, this strategy was unsuccessful. The second strategy, which worked, involved performing a partial digestion, as one of the restriction enzyme sites was located within the NFAT insert itself. We then performed directional cloning into a new vector containing the Flag tag .
| None |