Pgl3 basic luciferase reporter construct

Human IL2RA promoter (chr10:6,104,115–6,104,700), 1 kb intronic region containing rs12722489 (chr10:6,101,364–6,102,272) and a control region from STAT3 gene (chr17:40508494–40509570; all genomic coordinates are given for GRCh37/hg19 assembly) were amplified by PCR using genomic DNA from Jurkat cells as a template and specific primers containing the indicated cloning sites:
Promoter:
5’-ATATAAGCTTGCTGCCTGACCAGAATCTTG (HindIII),
5’-ATCCATGGCTTCCTGACCCTTGGGAC (NcoI)
Intronic region:
5’-AAGGATCCGCTGTACCCAGTGCGTAG (BamHI),
5’-TATGTCGACTACTGCAAAGTGGCTATGAAG (SalI)
Control:
5’-AGGATCCGGATTACAGGTGTATTTCACCAT (BamHI),
5’-TATGTCGACGTTGATGTAATTCCTTTAAATCTAT (SalI).
The A variant of rs12722489 was introduced into the 1 kb intronic fragment by overlap extension PCR using the following mutation-introducing primers: 5’-AAGGATCTGAGTGGTCTTGGAGG and 5’-CACTCAGATCCTTGGATAAGTCAC.
The IL2RA promoter was cloned into pGL3-basic luciferase reporter construct (Promega) using HindIII/NcoI restriction sites. Then 1 kb putative enhancer sequences were cloned immediately downstream of the luciferase gene using restriction sites indicated above. All constructs were verified by Sanger sequencing.
Control ERE-LUC reporter plasmid was kindly provided by Dr. George Reid and Dr. Frank Gannon [19 (link)].

The human cxcr5 promoter (−455/+368) and enhancer element (+2991/+5107) were amplified by PCR using genomic DNA from MCF-7 cells as a template and specific primers (see Supplementary Table 1) containing cloning sites. CXCR5 promoter variants containing deletions and mutations in the predicted NFκB sites were generated by two-step PCR mutagenesis and verified by sequencing. All variants of CXCR5 promoter were digested with HindIII and NcoI, cloned into pGL3-basic luciferase reporter construct (Promega, Madison, USA), and sequenced. Predicted cxcr5 gene enhancer element was cloned downstream of the luciferase gene using BamHI and SalI restriction sites.
NFκB response element consisting of 5 tandem NFκB consensus sites: GAG CTC GGG AAC TTC CGG GAA TTT CCG GGG AAG TCC GGG AAA TTC CGG GAC TTC CCC CCG GG, and minimal CMV promoter47 amplified from pEGFP-N3 plasmid (Clontech Laboratories, Madison, USA) with primers represented in Supplementary Table 1, were cloned into pGL3-basic luciferase reporter vector (Promega, Madison, USA) using restriction sites XhoI/HindIII and HindIII/NcoI respectively. Hyperactivation or inhibition of NFκB in MCF-7 cells was achieved using co-transfection of plasmid vectors expressing p65/RelA or dominant negative IκBα mutant48 (link).