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Example sentences for: pcmv
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Oligonucleotide primers for the PCR reaction were based on the published sequence of murine Rab24 [ 24 ] . Kpn1 and BamH1 restriction sites were added to the 5' and 3' primers respectively, to facilitate cloning of the PCR product in frame with a 5' sequence cassette encoding a myc epitope (EQKLISEEDL), previously introduced into the pCMV5 expression vector [ 78 ] . The Rab24 cDNA was altered by overlap-extension PCR [ 79 ] , using appropriate mutator oligonucleotides and Pfu DNA polymerase (Stratagene Inc., La Jolla, CA), so as to introduce amino acid substitutions at D123 (D123I), T120 (T120A), S22 (S22N), S67 (S67Q), and a deletion of residues 127 through 130 (ΔEDRR).
pCMV5 expression vectors containing Rab24 and Rab1B were engineered by overlap-extension PCR to encode the following chimeric proteins: Rab24/Rab1B, consisting of residues 1-165 of Rab24 [ 24 ] fused to residues 163-201 from Rab1B [ 80 ] ; Rab24(D123I)/Rab1B, consisting of residues 1-165 of Rab24 with the D123I substitution, fused to residues 163-201 of Rab1B; Rab1B/Rab24, consisting of residues 1-162 of Rab1B fused to residues 166-203 of Rab24; and Rab1B(N121I)/Rab24, consisting of residues 1-162 of Rab1B with the N121I substitution, fused to residues 166-203 of Rab24.
Other pCMV5 expression vectors encoding Rab1B(N121I) [ 28 ] , Rab6(N126I) and Rab8(N121I) [ 32 ] have been described in the indicated references.
Cells were transfected with pCMV5 expression vectors encoding mycRab24(D123I) as described earlier.
A reporter construct containing the basal promoter of the rat RIIβ 5'-flanking region (-394 to -123) upstream of a CAT-reporter gene (pCATbasic; Promega, Madison, WI) was used for assaying RIIβ promoter activity [ 18 ] . Expression vectors for USF1, USF2a and USF2b inserted 3' of the CMV promoter in pCR™3 (Invitrogen, Groningen, The Netherlands) were used to overexpress USF isoforms [ 12 ] . C/EBPβ LAP (full-length) and LIP (DNA-binding domain) expression vectors created in pCMV™3 (Stratagene, La Jolla, CA) were appreciated gifts from Dr. Shizua Akira (Hyogo College of Medicine, Hyogo, Japan).
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