A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene

A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn...

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Bibliographic Details
Published in:Gene 1996-02, Vol.169 (1), p.131-132
Main Authors: Yazynin, Sergey A., Deyev, Sergey M., Jucovi, Milan, Hartley, Robert W.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacterial Proteins
Barnase
barstar
Base Sequence
Cloning, Molecular - methods
Escherichia coli
Genes, Lethal
Genetic Vectors
Molecular Sequence Data
Plasmids
polylinker
Restriction Mapping
ribonuclease
Ribonucleases - metabolism
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Summary:A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhibitor, barstar (Bars), placed on the same plasmid. Full expression, however, is lethal. Induction is also lethal with the derived plasmid, pMT440, which has the pUC19 MCS inserted into barn. Under inducing conditions, transformation by the vector is lethal unless the product of the modified barn is inactivated by insertion of cloned DNA fragments into the MCS. Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec
ISSN:0378-1119
1879-0038
DOI:10.1016/0378-1119(95)00814-4