pGL3-Basic (Promega)
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). The DR-1X3 through DR-5X3 reporters were made with complimentary primers that were annealed and blunt-end–ligated into the Sma1 site upstream of the TK promoter.
DR-1X3 |
|
| Forward | TCAGTTCACAGTTCACAGTTCACAGTTCACAGTTCACAGTTCAGA |
| Reverse | TCTGAACTGTGAACTGTGAACTGTGAACTGTGAACTGTGAACTGA |
| DR-2X3 | |
| Forward | TCAGTTCAGCAGTTCAGCAGTTCAGCAGTTCAGCAGTTCAGCAGTTCAGA |
| Reverse | TCTGAACTGCTGAACTGCTGAACTGCTGAACTGCTGAACTGCTGAACTGA |
| DR-3X3 | |
| Forward | TCAGTTCAGGCAGTTCAGGCAGTTCAGGCAGTTCAGGCAGTTCAGGCAGTTCAGA |
| Reverse | TCTGAACTGCCTGAACTGCCTGAACTGCCTGAACTGCCTGAACTGCCTGAACTGA |
| DR-4X3 | |
| Forward | GATCAGTTCATGGCAGTTCATGGCAGTTCATGGCAGTTCATGGCAGTTCATGGCAGTTCAGATC |
| Reverse | GATCTGAACTGCCATGAACTGCCATGAACTGCCATGAACTGCCATGAACTGCCATGAACTGATC |
| DR-5X3 | |
| Forward | TCAGTTCACTGGCAGTTCACTGGCAGTTCACTGGCAGTTCACTGGCAGTTCACTGGCAGTTCAGA |
| Reverse | TCTGAACTGCCAGTGAACTGCCAGTGAACTGCCAGTGAACTGCCAGTGAACTGCCAGTGAACTGA |
| 2B6-XREM-PBREMa | |
| Forward | GATCGGTACCAGACTGTGCCAGATTGCACAACAC |
| Reverse | GATCGCTAGCCCACGAGGAGAGGACCAACAAAG |
| 3A4-XREM-pER6b | |
| pER6 Forward | GATCGAATTCTAAGAACCCAGAACCCTTGGAC |
| pER6 Reverse | GATCCTCGAGTGTGCTCTGCCTGCAGTTGGAA |
| XREM Forward | GATCGGTACCGTCCCAATTAAAGGTCATAAAG |
| XREM Reverse | GATCGAATTCCTCGTCAACAGGTTAAAGGAG |
| PBREM |
c
|
↵ a A polymerase chain reaction amplicon was generated from human genomic DNA that contained the 2B6 XREM sequences recently described (Wang et al., 2003a). 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. 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 thymidine kinase promoter.
↵ c The PBREM-TK-Luc reporter was described previously (Auerbach et al., 2003).