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Plasmid vector construction

Assignment 4

Below are restriction enzyme and features maps for two plasmids. The pGEM 4 has a T7 site that will initiation transcription through the multiple cloning site (POLYLINKER). We need to engineer a T7 RNA polymerase termination to the left of the multiple cloning site (POLYLINKER). The plasmid pGEMEX-2 has such a site called T7 TERM. Design a cloning strategy to transfer the T7 TERMination site from the pGEMEX-2 plasmid to the pGEM-4 plasmid to a location inbetween the POLYLINKER and the ampicillin resistance gene (APr). You need to keep the termination site in the same orientation relative to the MCS and ampicillin resistance gene. Find two possible cloning stategies and indicate which would be the best. To determine which is the best, take into consideration compatable overhangs vs blunt end ligations, single vs multiple fragments to insert into target site, ambiguities in the cut sites,