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Transforming genes coding for selectable functions, such as adenine phosphoribosyltransferase or thymidine kinase, have now been isolated by utilizing transformation in concert with molecular cloning.
Hirst has discussed the various aspects of structural soundness (e.g., folding stability or speed) that are singled out as proxies for selectable function [9].
Standard genetic methods were used for the construction of new strains and the presence of specific alleles was determined by a combination of phenotyping (e.g., for selectable markers), PCR diagnostics (e.g., for mating type), plus restriction mapping and DNA sequence analyses of PCR products (e.g., for alleles encoding epitope tags) [12], [53], [54].
However, this strategy only works for selectable traits.
We have developed mutant loxP vectors for selectable marker recycling and conditional knock-outs.
7 For selectable drug-resistance markers, enzyme and fluorescent protein reporters, etc; see http://ancillary.toxodb.org/docs/TransgeneReporters.seq.seq
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For screening for loss of selectable marker a different 3' primer for each transgene was used in combination with the 5' genotyping primer.
Genetically engineered microbes are of high practical importance due to their cost-effective production of valuable metabolites and enzymes, and the search for new selectable markers for genetic manipulation is of particular interest.
This demonstrated the potential for homozygous screening for a selectable phenotype in ES cells, but also highlighted the insertion bias of retroviral mutagenesis.
See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF.
See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp and the b9 ORF.
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