PCR products were cloned into polylinker
sites of pBI121 vectors to generate 35S::SEB-GUS and 35S::TetC-GUS constructs .
The expression vector consists of MBP preceded by methionine, with the final four amino acids replaced by 23 residues encoded by the pMAL-c2 polylinker
. The MBP protein was purified with affinity chromatography on amylose resin, as described in previous reports .
To facilitate protein purification, a 6xHis tag was added before MBP and a TEV protease cleavage site was added between MBP and polylinker
. The resultant plasmid was denominated by plou3-gsiA.
extension at the C-terminus (TPGRPAAKLN) corresponding to the fragment of the pQE30 plasmid polylinker
It includes different site of integration between interest gene and Pichia genome, variability of different expression host cell to secrete and express the recombinant enzyme, and different sites in the polylinker
for inserting foreign genes .
PCR products were digested with HindIII and EcoRV and cloned into the polylinker
region of the mammalian cell expression vector pcDNA3 (Invitrogen, San Diego, CA).
Subsequent sequencing of the 3' end of the insert and the flanking genomic DNA indicated that the polylinker
sequence containing a HindIII site was truncated by 10 bp.
-- Plasmids pO.380, a vector which contains 3.8 kb of Pu DNA subcloned into the EcoRI restriction site within the polylinker
region of pBluescribe (Stratagene) (McLean et al.
This 812-bp linked sequence was cloned into the polylinker
region of the pSP64polyA plasmid.
sites permitted directional cloning into single-stranded M13 bacteriophages (Medlin et al., 1988) for the dideoxynucleotide-mediated chain-termination sequencing protocol (Sanger et al., 1977).
The forward primer for vector amplification (57-CACCACCACCACCACCACTGAGAT-37) matched to the 3' side of the polylinker
region of expression vector pET28a.