Cloning and/or sequencing vector
First Claim
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1. A method for propagating a cloning or sequencing vector that lacks an insert, said method comprising:
- introducing said vector into a first prokaryotic cell, said cloning vector comprising a promoter operably linked to a nucleotide sequence encoding a CcdB poison protein, said cell expressing a CcdA antidote protein;
propagating said vector in said first prokaryotic cell, thereby expressing said CcdB poison protein without killing the cell; and
recovering the vector.
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Abstract
A cloning and/or sequencing vector enables recombinant clones to be selected directly. The vector encodes a fusion protein which includes a protein poison.
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Citations
18 Claims
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1. A method for propagating a cloning or sequencing vector that lacks an insert, said method comprising:
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introducing said vector into a first prokaryotic cell, said cloning vector comprising a promoter operably linked to a nucleotide sequence encoding a CcdB poison protein, said cell expressing a CcdA antidote protein; propagating said vector in said first prokaryotic cell, thereby expressing said CcdB poison protein without killing the cell; and recovering the vector. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of growing prokaryotic cells that comprise a cloning vector expressing a CcdB poison protein without selecting for prokaryotic cells containing mutations in the ccdB gene, comprising:
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providing the prokaryotic cells with a gene encoding a CcdA antidote protein so as to antagonize the activity of the CcdB poison protein providing said cells with a gene encoding E. coli GyrA having a mutation at amino acid position 462; and growing said cells while expressing the CcdB poison protein. - View Dependent Claims (8)
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9. A method of growing prokaryotic cells that comprise a cloning vector encoding a CcdB poison protein comprising:
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providing said prokaryotic cells with a cloning vector with a pUC origin of replication, said cloning vector also encoding the CcdB poison protein; and providing said prokaryotic cells with E. coli GyrA having a mutation at amino acid position 462. - View Dependent Claims (10, 11)
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12. A method for propagating a cloning or sequencing vector that lacks an insert, said method comprising:
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introducing said vector into a first prokaryotic cell, said cloning vector comprising a promoter operably linked to a nucleotide sequence encoding a CcdB poison protein, said cell expressing a polypeptide consisting of the 41 -carboxy terminal amino acids of a CcdA antidote protein; propagating said vector in said first prokaryotic cell, thereby expressing the CcdB poison protein without killing the cell; and recovering the vector. - View Dependent Claims (13, 14, 15, 16, 17, 18)
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Specification