A ori B
 Cm' L 4
 Sph| HA tag


 pTAM34
 pTAM35 t
 6.9 kb I~H) SD~td~h
 (b+7-hi SD Ofl (b-y+AdSh





 Diplest
 Sph|



 Vector Insert
 3 1 kb Ligate 623 bp


 C HA tag
 rp SD b,


 pTA M40
 3.7 kb SD
 Sph|
 (bH -~ _HA & +7-his





Figure 2-2. Construction of the single transcript expression system. A) A unique
 restriction enzyme site, Sphl, was created in conjunction with the histidine tag
 between the Shine Dalgarno sequence and the first codon of uncF(b) to create
 pTAM3. B) An Sphl site was added to pTAM34 downstream of the HA-
 tagged b subunit and behind another favorable Shine Dalgarno sequence. The
 two plasmids were digested with Sphl and BstEII. The 3.2 kb vector fragment
 from pTAM34 and the 623 bp insert fragment from pTAM3 5 were ligated.
 Additional mutagenesis (see Materials and Methods) resulted in C) a 3.7 kb
 plasmid, pTAM40, which expressed bA7-HA and b+7-his from the same promoter
 and included the ampicillin resistance gene and the pUC 18 origin of
 replication. In similar constructions, plasmids pTAM41 (bwt-HA and b+7-his)
 and pTAM42 (bwt-HA and bwt-his) were created.