For generating of the human CHAC1 promoter reporter construct, genomic DNA from HEK293 cells was extracted, and the human CHAC1 promoter (−265/+77) was amplified using polymerase chain reaction (PCR). The PCR product was then cloned into the pGL3-Basic (pGL3b) vector (Promega). Other constructs containing either deleted or mutated human CHAC1 promoters were also prepared by PCR and sequence specific mutagenesis. The promoter region was defined using the NCBI Reference Sequence file NM_024111, and our prior reports [36 (link),39 (link)]. For generating mouse CHOP, cDNA was amplified from C/57BL6 mouse by reverse transcriptional-polymerase chain reaction (RT-PCR) and then cloned into the pcDNA3.1 vector (Life Technologies). Reporter constructs for the mouse CHAC1 promoter and the ATF4 expression vector were previously described [37 (link)].
Pgl3 basic pgl3b vector
Manufactured by Promega
The PGL3-Basic (pGL3b) vector is a plasmid DNA construct used as a backbone for cloning and expressing genes of interest in various cell types. It contains a firefly luciferase gene and a multiple cloning site for inserting the gene of interest. The vector can be used to assess gene expression levels by measuring the luminescent signal produced by the luciferase enzyme.
Automatically generated - may contain errors
2 protocols using pgl3 basic pgl3b vector
1
Generation of CHAC1 Promoter Constructs
2
Cloning and Mutagenesis of AE4 Promoter
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The promoter region of the AE4 gene22 (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-Foxi344 (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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