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New cosmid vectors developed for eukaryotic DNA cloning
Authors:G Brady  H M Jantzen  H U Bernard  R Brown  G Schütz  T Hashimoto-Gotoh
Institution:Institute for Cell and Tumor Biology, German Cancer Research Center, Im Neuenheimer Feld, 280, D-6900 HeidelbergF.R.G. Tel. 6221-48-4411
Abstract:A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon Matthias et al., EMBO J. 2 (1983) 1487-1492].
Keywords:Recombinant DNA  G418 selection  DNA mediated gene transfer  shuttle vector  cosmid rescue  Ap  ampicillin  BPVI  bovine papilloma virus  type I  Cm  chloramphenicol  FTL  freeze thaw lysate  kb  kilobase pairs  Km  kanamycin  LS  low salt medium  MAC  maximal allowance concentration  SE  sonic extract  Sm  streptomycin  Tc  tetracycline
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