A bacteriophage T7-based expression vector, pBT7, with color selection for the recombinant.
A bacterial expression plasmid, pBT7, based on a bacteriophage T7 RNA polymerase/promoter system, has been devised for positive color selection of clones containing an insert. The T7 gene 10 promoter and its N-terminal portion, 89 nucleotides in length, have been amplified by the polymerase chain reaction and cloned into the multiple cloning site of plasmid pUC18 in the opposite direction to lacZ'. This insertion does not inactivate the lacZ alpha-peptide activity. Hence, bacteria carrying pBT7 without an insert form blue colonies on an indicator plate, while bacteria carrying recombinant clones with an insert appear as white colonies. Using pBT7, part of the Caenorhabditis elegans unc-13 gene product has been overproduced at a high level, approaching 50% of total Escherichia coli protein.
Maruyama
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Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037.
Mikawa
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YG
Maruyama
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HI
D000818:Animals; D017123:Bacteriophage T7; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics; D007202:Indicators and Reagents; D007763:Lac Operon; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009841:Oligonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C; D011955:Receptors, Drug / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D012394:Rosaniline Dyes
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D015801:Helminth Proteins; D007202:Indicators and Reagents; D009841:Oligonucleotides; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; C048139:Coomassie blue; D011493:Protein Kinase C