and introduce the ecorv restriction site the resulting pcr product was cloned into pgem t easy promega yielding vector pxh0302 as a next step in the construction of pxhsm45n pmh54 was treated with the restriction enzymes sse8387i and ecorv and the resulting fragment was cloned into the ecorv site of pxh0302 after being blunted by t4 dna polymerase treatment yielding vector pxh0902 after excision of the sse8387i ecorv fragment of pmh54 the remaining vector was also blunted by t4 dna polymerase treatment and religated resulting in plasmid pxh1401 finally plasmid pxh0902 was treated with restriction enzymes nhei and bsshii and the resulting fragment was cloned into pxh1401 treated with the same enzymes yielding pxhsm45n for the construction of pxhmsmn first the orfs 4 5 and m were removed from pmh54 by restriction of this vector with enzymes sse8387i and bsshii followed by treatment with t4 dna polymerase and religation of the remaining vector which yielded pxh u25a1 45m5 u2032 next the fragment resulting from treatment of pxh0302 with enzymes nhei and bsshii was cloned into pmh54 treated with the same enzymes resulting in pxhmen subsequently the fragment obtained after restriction of pxhmen with ecorv was cloned into pxh1802 described above which was digested with hindiii and treated with klenow fragment of dna polymerase i yielding pxh0305b the fragment obtained by restriction of pmh54 with enzymes mlui and ecorv was cloned into pb59 2 treated with the same enzymes which resulted in vector pxh2801 next the fragment resulting from the treatment of pxh2801 with restriction enzymes kpni and psti.