Restriction digests of the clone give the following sizes (kb): KpnI--5.8; ApaI--5.8; HindIII--5.8. Preparation of the vector for cloning includes linearization with ApaI, gel purification of the linearized vector, and treatment with T4 DNA polymerase in the presence of dATP. The target sequence can be amplified using sequence specific primers modified at the 5' end to contain an additional 13 nt complementary to the vector. The forward primer should contain 5'-CCTGCTCGTCTGA-3' followed by 12-15 nt target-specific sequence. The reverse primer should contain 5'-GGTGGTGCTCTGA-3' followed by 12-15 nt target-specific sequence. The amplified sequence should be gel purified and treated with T4 DNA polymerase in the presence of dTTP. Annealing of the vector and the amplification product forms a duplex that can be used directly for transformation. Sequences amplified using these primers are also compatible with the pCAT/LIC vector (ATCC 87046). Ligation-independent promoter-cloning vector, containing the luciferase coding sequence downstream from the cloning site. The order of the major features in the plasmid is: pMB1 ori - ampR - f1 ori - KpnI - ApaI - HindIII - luciferase coding sequence - SV40 splice site and polyadenylation. |
