pGL3-Basic (Promega, Madison, WI) clones The pGL3 basic vector was engineered with the thymidine kinase core promoter as described previously to generate a TK-luc reporter (Auerbach et al., 2003). DR-1X3 through DR-5X3 reporters were made with complimentary primers following their annealing and blunt end ligation into the Sma1 site upstream of TK promoter.
| Name | Primers |
|---|---|
| DR-1X3 | FP: TCAGTTCACAGTTCACAGTTCACAGTTCACAGTTCACAGTTCAGA |
| RP: TCTGAACTGTGAACTGTGAACTGTGAACTGTGAACTGTGAACTGA | |
| DR-2X3 | FP: TCAGTTCAGCAGTTCAGCAGTTCAGCAGTTCAGCAGTTCAGCAGTTCAGA |
| RP: TCTGAACTGCTGAACTGCTGAACTGCTGAACTGCTGAACTGCTGAACTGA | |
| DR-3X3 | FP: TCAGTTCAGGCAGTTCAGGCAGTTCAGGCAGTTCAGGCAGTTCAGGCAGTTCAGA |
| RP: TCTGAACTGCCTGAACTGCCTGAACTGCCTGAACTGCCTGAACTGCCTGAACTGA | |
| DR-4X3 | FP: GATCAGTTCATGGCAGTTCATGGCAGTTCATGGCAGTTCATGGCAGTTCATGGCAGTTCAGATC |
| RP: GATCTGA CTGCCATGAACTGCCATGAACTGCCATGAACTGCCATGAACTGCCATGAACTGATC | |
| DR-5X3 | FP: TCAGTTCACTGGCAGTTCACTGGCAGTTCACTGGCAGTTCACTGGCAGTTCACTGGCAGTTCAGA |
| RP: TCTGAACTGCCAGTGAACTGCCAGTGAACTGCCAGTGAACTGCCAGTGAACTGCCAGTGAACTGA | |
| 2B6-XREM-PBREMa | FP: GATCGGTACCAGACTGTGCCAGATTGCACAACAC |
| RP: GATCGCTAGCCCACGAGGAGAGGACCAACAAAG | |
| 3A4-XREM-pER6b | pER6FP: GATCGAATTCTAAGAACCCAGAACCCTTGGAC |
| pER6RP: GATCCTCGAGTGTGCTCTGCCTGCAGTTGGAA | |
| XREMFP: GATCGGTACCGTCCCAATTAAAGGTCATAAAG | |
| XREMRP: GATCGAATTCCTCGTCAACAGGTTAAAGGAG |
FP, forward primer; RP, reverse primer.
↵ a The PBREM-TK-Luc reporter was described previously (Auerbach et al., 2003). A PCR amplicon was generated from human genomic DNA that contained the 2B6 XREM sequences recently described (Wang et al., 2003a). The PCR primers are shown. The amplicon was ligated upstream of the TK promoter using the KpnI and NheI restriction sites
↵ b Amplicons encompassing the proximal (p) ER-6 (Barwick et al., 1996) and distal XREM (Goodwin et al., 1999) sequences in the CYP3A4 promoter were amplified separately with the primers shown. Individual amplicons were digested with EcoRI, purified, and ligated. The ligation was then amplified with the XREMFP and pER6RP. The product from this second amplification was then blunt end ligated into the SmaI site upstream of the TK promoter