Restriction digests of the clone give the following sizes (kb): BamHI--4.1; EcoRI--2.8, 0.78, 0.44; HindIII--4.1; BstEII--4.1. This was constructed from pXF3 (pBR322 minus the poison sequence), a SV40 ori, and a rat preproinsulin genomic sequence under the control of RSV LTR (pBC81delta4 cut at BgIII and BalI and inserted at the BamHI and ClaI sites). cDNA sequences with a translation initiation codon are usually inserted between the unique HindIII and BamHI sites (5' to 3'). Polyadenylation signals (and possibly carboxy terminal amino acids) are contributed by the preproinsulin gene. Genomic sequences are normally inserted between unique HindIII and SmaI sites (5' to 3'). This deletes the preproinsulin gene from the vector. cDNA clones without an initiation codon may be cloned in frame into the unique BamHI site 3' to the AUG of the rat preproinsulin gene. This results in 5 - 6 additional amino terminal amino acids from the insulin signal peptide. |
