Title: The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope Document date: 1992_12_2
ID: vznqgnzd_14
Snippet: Deletion of the TM and the CT of gp210 was achieved by inserting a stop codon after nucleotide 5,424 into the gp210:HA cDNA. This was accomplished by inserting a PCR product into a unique BgllI site at nucleotide 5,139. This PCR product is identical to the gp210 eDNA from the BglH site downstream to the codon for amino acid residue 1,808, after which a stop eodon is inserted. It was synthesized using the eDNA clone 49-10 as a template and two oli.....
Document: Deletion of the TM and the CT of gp210 was achieved by inserting a stop codon after nucleotide 5,424 into the gp210:HA cDNA. This was accomplished by inserting a PCR product into a unique BgllI site at nucleotide 5,139. This PCR product is identical to the gp210 eDNA from the BglH site downstream to the codon for amino acid residue 1,808, after which a stop eodon is inserted. It was synthesized using the eDNA clone 49-10 as a template and two oligonucleotide primers, a sense primer encoding nucleotides 5,125-5,141 and an antisense primer encoding nncleotides 5,409-5,424 followed by an in-frame stop codon and a 5' BglII linker. After cleavage with BglII, the PCR product was introduced into the gp210:HA reading frame at the BgllI site. The orientation and the reading frame of the insert were determined by DNA sequencing. This gp210-(TM + CT):HA mutant (see Fig. 1 ) was inserted into the pSVL plasmid for expression studies.
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