The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane

p58AN,TMI+2. A fragment containing codons 280 through 637 and the 3' untranslated region was amplified from p58FL by PCR using a 5' PCR primer, 5'-GAGAATTCGGTACCAATCCAAGGAAGC'IGCAC-3' (Kpni site is underlined) and 3' PCR primer, 5'-CTGAATTCTCTAGAAGGTCA-TAGACACTCAGT-3' (XbaI site is underlined). The resulting fragment was digested with KpnI and XbaI and used to replace the KpnI-XbaI fragment (containing codons 45 through 206) of p58ATM1-8, p58AN,TMI+2 conrains p58 amino acids 1-45 + 280-637. Next, a fragment containing the complete amino terminal domain was amplified from p58ATM2-8 by PCR using a 5' PCR primer, 5~ ' and 3' PCR primer 5'-~TACCTCTICCACCAAATTCTAC-3' (KpnI site is underlined). The resulting fragment was digested with KpnI and the fragment containing codons 45 through 205 was ligated into the unique KpnI site of p58AN,TMl+2. The correct orientation was determined by restriction digest analysis, p58ATMl+2 contains amino acids 1-205, followed by a glycine and threonine (introduced by the cloning), followed by amino acids 280-637.