Simultaneous cloning of open reading frames into several different expression vectors
نویسندگان
چکیده
منابع مشابه
Simultaneous cloning of open reading frames into several different expression vectors.
Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into exp...
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A plasmid was constructed that facilitates the cloning and expression of open reading frame DNA. A DNA fragment containing a bacterial promoter and the amino terminus of the cI gene of bacteriophage lambda was fused to an amino-terminally deleted version of the lacZ gene. An appropriate cloning site was inserted between these two fragments such that a frameshift mutation was introduced upstream...
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The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fr...
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We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression. By varying the base sequence preceding the uORF, we sought to vary the translation initiation rat...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 2003
ISSN: 0736-6205,1940-9818
DOI: 10.2144/03353st05