Restriction digests of the clone give the following sizes (kb): EcoRI--4.9; BglII--4.9; XbaI--4.9; BamHI--4.9; EcoRI/HindIII--3.4, 1.5; EcoRI/BglII--3.4, 1.5. A shuttle expression vector permitting rapid cloning of putative intracellular targeting sequences as N-terminal, C-terminal, and/or internal fusions with the normally cytosolic chicken muscle pyruvate kinase (PKM2). Permits one step subcloning into any of the pJ(X)omega mammalian expression vectors (ATCC 37719-37724) for versatile promoter control, and into the pGE(X) bacterial fusion protein purification system. The PKM2 sequence encodes amino acids 17-476. The PKM2 coding region contains a unique SalI site. The BamHI site consists of 2 engineered codons added to the end of the PKM2 sequence and immediately followed by an in-frame TGA stop codon. The other 3' sites are outside the reading frame. The order of the major features in the plasmid is: pMB1 ori - SV40 ori - SV40 early promoter - HindIII - Kozak/ translation start sequence - BglII - PKM2 sequences - translation stop - BamHI - XbaI - EcoRI - ClaI - SV40 polyadenylation - ampR. |
