Cloning and Mutagenesis of AE4 Promoter

The promoter region of the AE4 gene^{22 (link)} was amplified by PCR from mouse genomic DNA and cloned into the pGL3-Basic (pGL3b) vector (Promega). pGL3b-AE4 mutant constructs were generated by site directed mutagenesis of one or both TTT > GGG sequences in the promoter. N-terminal and C-terminal truncations of Foxi3 as well as full length (FL; 1–399) Foxi3 were amplified from pCMV-SPORT6-Foxi3^{44 (link)} and cloned in a 3XFLAG vector (Sigma). A Foxi3 FL NLS mutant construct was generated by site directed mutagenesis of the predicted nuclear localization sequence (NLS) in 3xFLAG Foxi3 FL. C-terminal Foxi3 fragments were amplified and cloned as a fusion construct with the DNA binding domain of GAL4 in pBIND vector (Promega). 9aaTAD Mut1 and 2 were generated by site directed mutagenesis of the predicted 9aaTAD in pBIND Foxi3 350–399 construct. PPP2R2A, PPP2CB, PPP2R1B was amplified by PCR using mouse cDNA and cloned in pCMV-Myc vector (Clontech). All cloning was performed with an In-Fusion HD Cloning kit (Clontech).

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Singh S., Jangid R.K., Crowder A, & Groves A.K. (2018). Foxi3 transcription factor activity is mediated by a C-terminal transactivation domain and regulated by the Protein Phosphatase 2A (PP2A) complex. Scientific Reports, 8, 17249.

Publication 2018

pGL3-Basic (pGL3b) vector pBIND vector pCMV-Myc vector In-Fusion HD Cloning kit

Cdna Genomic Mouse Pgl3 Ppp2cb Ppp2r1b Promega Site directed mutagenesis Vector

Corresponding Organization :

Other organizations : Baylor College of Medicine

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Variable analysis

independent variables

One or both TTT > GGG sequences in the promoter of the AE4 gene
N-terminal and C-terminal truncations of Foxi3
Full length (FL; 1–399) Foxi3
Foxi3 FL NLS mutant construct
9aaTAD Mut1 and 2 in pBIND Foxi3 350–399 construct

dependent variables

Not explicitly mentioned

control variables

Mouse genomic DNA
PGL3-Basic (pGL3b) vector (Promega)
PCMV-SPORT6-Foxi3
3XFLAG vector (Sigma)
PBIND vector (Promega)
PCMV-Myc vector (Clontech)
All cloning performed with an In-Fusion HD Cloning kit (Clontech)

controls

No positive or negative controls were explicitly mentioned

Annotations

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