Patent ID: 6265218
Filing Date: 2001-07-24
Classification: A61K,A61P,C07K,C12N

Abstract:
A method for making a plasmid comprising a DNA sequence encoding a product comprising:(i) introducing a heterologous DNA sequence encoding a product operatively linked to a promoter into a plasmid, said plasmid further comprising a DNA sequence encoding an antibiotic resistance selection marker operatively linked to a promoter and wherein restriction enzyme cleave sites separate the two DNA sequences;(ii) amplifying said plasmid;(iii) cleaving by restriction enzymes said amplified plasmid to form two linear fragments, the first of which comprises said heterologous DNA sequence operatively linked to a promoter, the second of which comprises said DNA sequence encoding said selection marker operatively linked to a promoter;(iv) circularizing each of said fragments to form two plasmids, the first of which comprises said heterologous DNA sequence operatively linked to a promoter and lacking said DNA sequence encoding a selection marker, the second of which comprises said DNA sequence encoding said selection marker operatively linked to a promoter and lacking said heterologous DNA sequence;(v) separating the plasmids obtained in (iv); and(vi) isolating the plasmid comprising the heterologous DNA sequence encoding a product operatively linked to a promoter.