Restriction digests of the clone give the following sizes (kb): EcoRI/HindIII--2.6, 1.7, 1.0, 0.3; HindIII--2.9, 2.7; EcoRI--4.2, 1.4; BglII--5.6. A EcoRI/HindIII digest gives fragments of 2.63, 1.68, 0.97 and 0.32 kb. The SV40 promoter has a 3 bp deletion at the BglI site within the origin of replication to prevent autonomous replication in the presence of SV40 large T-antigen. The sequence of the oligonucleotide is AGCTTGGTAC CATGGACCTC GAGGCCTAGC TAGCTAG GAGCT. Kanamycin resistance should be checked at regular intervals. This is best done during amplification steps using LB medium supplemented with 50 ug/ml ampicillin and 12.5 ug/ml kanamycin. One of 3 shuttle expression vectors (pMSEx-1-3, ATCC 37835-37837) containing a synthetic oligonucleotide providing a translation initiation signal, followed by a StuI site in 3 different positions, followed by a stop signal in all reading frames. The MSV LTR provides a polyadenylation signal as well as an enhancer for the P1/thymidine kinase promoter regulating the Tn5 kanR/G418R transcription unit. The order of the major features in this plasmid is: SV40 promoter - oligonucleotide/StuI/oligonucleotide - MLV-LTR - P1 promoter - TK promoter - kanR/G418R - TK polyadenylation signal - ampR. |
