EP 68740 B1

Actual granted independent claims

EP 68740 B1

Claim 1Plasmid pKC222 which contains the 2.75 kb Sal I/Bgl II restriction fragment of plasmid pKC203 as obtainable from E. coli JR225 ATCC 31912 ligated to the Sal I/Bgl II restriction fragment of plasmid pKC7, and which confers resistance to antibiotics ampicillin, hygromycin B and G418 when transformed into an E. coli cell.
Claim 2A recombinant DNA cloning vector that comprises a eukaryotic promoter, the plasmid pBR322 replicon, and

a) the 7.5 kb Bgl II restriction fragment of plasmid pKC203 that conveys resistance to antibiotics hygromycin B and G418; or
b) the 2.75 kb Bgl II/Sal I restriction fragment of plasmid pKC203 or plasmid pKC222 that conveys resistance to antibiotics hygromycin B and G418; or
c) the 1.51 kb Sac I/Bgl II restriction fragment of plasmid pKC222 that conveys resistance to antibiotic hygromycin B; or
d) the 1.65 kb Eco RI/Sal I restriction fragment of plasmid pKC222 that conveys resistance to antibiotic G418; or
e) a DNA sequence derived from the said 7.5 kb restriction fragment that conveys resistance to either or both antibiotics hygromycin B and G418; the resistance to antibiotics hygromycin B and/or G418 being conveyed when the vector is transformed into a host cell that is sensitive to one or both of said antibiotics, the host cell being susceptible to transformation, cell division, and culture; subject to the limitations

  • that the promoter is functional in mouse or yeast cells,
  • that the restriction fragments conveying antibiotic resistance are adjacent to and, in a eukaryotic host cell, transcribed from the promoter, and
  • that the DNA sequences conveying resistance to antibiotic G418 do not code for the enzyme phosphotransferase.
Claim 22Plasmid pSC701 obtained by self-ligation of the 7.3 kb Bgl II restriction fragment of plasmid pKC203 (ATCC 31912) and having the restriction map shown in Figure 6.
Claim 23Plasmid pKC257 obtained by incubating plasmid pSC701 with Hae II restriction enzyme, self-ligating the resulting mixture of restriction fragments, transforming an E. coli K12 strain with the ligated mixture, and screening the transformants for hygromycin B resistance; and having the molecular weight and restriction map shown in Figure 7.
Claim 24Plasmid pKC259 obtained by incubating plasmid pSC701 with Hae II restriction enzyme, self-ligating the resulting mixture of restriction fragments, transforming an E. coli K12 strain with the ligated mixture, and screening the transformants for ampicillin and hygromycin B resistance; and having the molecular weight and restriction map shown in Figure 7.
Claim 25Plasmid pKC261 obtained by self-ligation of the 3.2 kb Sau3A I restriction fragment of plasmid pKC257 and having the restriction map shown in Figure 7.
Claim 26Plasmid pKC275 obtained by ligating the 396 base plac containing Hae II restriction fragment of plasmid pUR222 as obtainable from E. coli K12 BE1166 NRRL B-15023 and a mixture of Hae II restriction fragments of plasmid pKC261, transforming and E. coli K12 strain with the ligated mixture, and selecting transformants containing only a 3.6 kb plasmid; and having the restriction map shown in Figure 8.
Claim 27Plasmid pKC264 obtained by ligating the 2 µ EcoR I restriction fragment of plasmid YEp24 as obtainable E. coli K12 BE1139 NRRL B-15022 and a mixture of EcoR I restriction fragments of plasmid pKC259, transforming an E. coli K12 strain with the ligated mixture, and selecting transformants containing an 7.2 kb hygromycin B, apramycin and G418 resistance-conferring plasmid; and having the restriction map shown in Figure 8.
Claim 28The 7.5 kb Bgl II restriction fragment of plasmid pKC203.
Claim 29The 2.75 kb Sal I/Bgl II restriction fragment of plasmid pKC203.
Claim 30The 1.51 kb Sac I/Bgl II restriction fragment of plasmid pKC222.
Claim 31The 1.65 kb EcoR I/Sal I restriction fragment of plasmid pKC222.