EP 135291 B1

EP 135291 B1
Claims for the states: BE, CH, DE, FR, GB, IT, LI, LU, NL, SE
Claim 1A DNA encoding the last 338, 339 or 349 amino acids of hygromycin B phosphotransferase.
Claim 15
Plasmid pIT123 which is shown in Figure 1 and constructed by the steps of: a) ligating the 2.75 kb Sal I/Bgl II fragment of plasmid pKC203 (ATCC Deposit No. 31912) and the 4.1 kb Sal I/Bgl II fragment of plasmid pKC7 (ATCC Deposit No. 37084) to form plasmid pKC222;
b) providing the 325 bp Hph I-Pst I fragment of plasmid pKC222 with a BamH I linker and an EcoR I linker;
c) ligating the linker provided fragment of step (b) with the EcoR I-BamH I digest of known plasmid pBR322 to provide plasmid pIT122; and
d) ligating the 1.45 kb EcoR I fragment of plasmid pKC222 and the EcoR I digest of plasmid pIT122.
EP 135291 B1
Claims for the state: AT
Claim 1
A process for preparing a plasmid comprising a DNA sequence encoding the last 338 amino acids of hygromycin B phosphotransferase, either alone or in translational reading phase with a transcriptional and translational activator sequence-containing gene or portion of a gene, which comprises the steps of: a) ligating the 2.75 kb Sal I/Bgl II fragment of plasmid pKC203 (ATCC Deposit No. 31912) and the 4.1 kb Sal I/Bgl II fragment of plasmid pKC7 (ATCC Deposit No. 37084) to form plasmid pKC222;
b) providing the 325 bp Hph I-Pst I fragment of plasmid pKC222 with a BamH I linker and an EcoR I linker;
c) ligating the linker provided fragment of step (b) with the EcoR I-BamH I digest of known plasmid pBR322 to provide plasmid pIT122; and
d) ligating the 1.45 kb EcoR I fragment of plasmid pKC222 and the EcoR I digest of plasmid pIT122, to obtain the plasmid pIT123 shown in Figure 1.
Claim 9
The process for preparing plasmid pIT207, shown in Figure 3, which comprises ligating the 750 bp BamH I-Bgl II fragment of plasmid pIT118 (NRRL deposit No. B-15441) into BamH I-digested plasmid pMC1587 (NRRL deposit No. B-15442).
Claim 11The process for preparing plasmid pIT143 which comprises: a) digesting the 958 bp Cla I-Hinc II fragment of plasmid pIT141 (NRRL deposit No. B-15602) with the restriction enzyme Mbo II;
b) removing the resultant extensions attaching BamH I linkers, and
c) ligating the linker-containing fragment into BamH I-digested plasmid pUC8.