Cloning vector containing marker inactivation system

Details Cloning vector containing marker inactivation system Cloning vector containing marker inactivation system

1998-05-01

2000-10-03

Guzo, David

Chemistry: molecular biology and microbiology

Vector, per se

435 29, 435 9141, 435471, C12N 1565, C12N 1566, C12Q 102

Patent

active

061271713

ABSTRACT:
Provided are lacZ.alpha. gene fragments which have been modified to introduce multiple restriction enzyme sites. Vectors according to the present invention include at least one promoter operatively linked to a DNA sequence encoding lacZ.alpha. (.alpha.-peptide); multiple cloning sites cleavable by distinct restriction enzymes which have been introduced within a lacZ coding sequence from and including the codon for amino acid 8, and in the lacZ coding sequence downstream of the codon for amino acid 8, in forming the modified lacZ.alpha. coding sequence; and a replicon. The vector may further comprise one or more additional features useful for protein expression and other molecular manipulations. Also provided are methods of using the vectors wherein a DNA molecule is cloned into at least one restriction enzyme site in the modified lacZ.alpha. coding sequence, in forming recombinant vectors; introducing the recombinant vectors into competent host cells; growing the host cells in the presence of a chromogenic substrate cleavable by .beta.-galactosidase; and screening for indicia of lac operon marker inactivation. The high accuracy of color selection afforded by the modified lacZ.alpha. coding sequence of the present invention provides a first opportunity at performing gap-free shotgun sequencing and development of ordered genomic libraries.

REFERENCES:
patent: 5474933 (1995-12-01), Weiner et al.
Heinrich et al. Positive-selection vector with enhanced lytic potential based on a variant of pX174 phage gene E. Gene 154: 51-54, Mar. 1995.
Shizuya et al. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89: 8794-8797, Sep. 1992.
Sambrook et al., 1989, "Molecular Cloning: A laboratory manual", second edition. Cold Spring Harbor Laboratory Press, pp. 1.8,1.9,1.13,1.14,1.53-1.59,1.85,1.86, and 4.7-4.11.
Zabin, "B-Galactodidase .alpha.-complementation", 1982, Molecular and Cellular Biochemistry, vol. 49, pp. 87-96.
Dunn et al., "Improved Peptide function from random mutagenesis over short 'windows'", 1988, Protein Engineering, vol. 2, No. 4, pp. 283-291.

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