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Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting...
Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27copies/cell) and high (∼240copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants...
A plasmid system for site-specific integration into and excision and recovery of gene constructs and lacZ gene fusions from the Escherichia coli chromosome was developed. Plasmid suicide vectors utilizing the origin of replication of R6K plasmids and containing the attP sequence of bacteriophage λ, multiple cloning site, and antibiotic resistance markers facilitate reversible integration into the...
A series of cloning vectors with conditional, temperature-sensitive replication that are selectable with ampicillin, chloramphenicol, and kanamycin has been constructed. These vectors are derivatives of a pSC101 mutant that can replicate only at low temperatures. The cloning vectors carry a number of unique restriction sites and provide for screening of recombinant plasmids by α complementation. These...
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