The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

Two additional PCR products were synthesized encoding the remaining two-thirds of the full-length eDNA. The middle segment was synthesized using the eDNA clone 47-11 as a template and oligonucleotide primers encoding nucleotides 1,937-2,016 (sense) and nucleotides 3,826-3,854 (antisense). The 3' segment was synthesized using the eDNA clone 49-10 as a template and oligonucleotide primers encoding nucleotides 3,845-3,873 (sense) and nucleotides 5,664-5,708 plus a 5' SalI site (antisense). Point mutations were introduced at nucleotide positions 1,992 and 3,849 of the above oligonucleotide primers to create two unique BstBI sites at positions 1,990 and 3,847 without altering the amino acid sequence of gp210. These sites were used to assemble a full-length gp210 eDNA with flanking, unique SaiI sites. Sequencing of this clone revealed four nucleotide changes produced during the PCR reactions. Two of these led to changes in the amino acid sequence, one at position 491 (serine to glycine) and the other at position 1,131 (leucine to histidine).