Pgl4.51 vector

A promoter–deletion experiment was designed to monitor promoter activity of the 5′-flanking region. DNA fragments with a series of nested deletions were generated, and five DNA fragments were inserted into the pGL4.10 vector and named pGL4-1 (−3000 to 0 bp), pGL4-2 (−2400 to 0 bp), pGL4-3 (−1800 to 0 bp), pGL4-4 (−1200 to 0 bp), and pGL4-5 (−600 to 0 bp). pGL4.51 vector (Promega, E132A) with a CMV promoter was used as a reference (positive control), while pGL4.10 vector (Promega, E665A) without promoter was used as negative control.
The pEGFP-N3 expression plasmid was purchased from Invitrogen. The tilapia gas2 open reading frame (ORF) was amplified by polymerase chain reaction (PCR), cloned into the pEGFP-N3 with the GAS-N-S1 and GAS-N-A1 primers (Additional file 1: Table S1), and named pEGFP-N3-GAS2.

Plasmid containing MDM2 minigene sequence was obtained from Dr. D. Chandler (Nationwide Children’s Hospital) (Singh et al. 2009 (link)). The Luc-MDM2 construct contains an MDM2 minigene sequence within the luciferase ORF in the pGL4.51 vector (Promega, Madison, WI, U.S.A.), which contains synthetic firefly luciferase luc2 gene under the control of CMV promoter. Intron 3 through Intron 11 of the MDM2 minigene sequence is inserted at nucleotide 477 of the luciferase ORF. A stop codon was engineered into exon 4 of MDM2.

An EGFP‐N1 vector (Clontech Laboratories, Mountain View, CA, USA) encoding the enhanced green fluorescent protein gene and a pGL4.51 vector (Promega Corporation, Madison, WI, US) encoding the luciferase gene were amplified in E. coli (DH5α) cells and then purified using an endotoxin‐free Plasmid Giga Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The pDNA stock solution was adjusted to 1 mg mL⁻¹ with UltraPure DNase/RNase‐Free distilled water (Thermo Fisher Scientific, Waltham, MA, USA).