integration site for streptomyces phage phibt1 and development of site-specific integrating vectors.despite extensive similarities between the genomes of the streptomyces temperate phages phic31 and phibt1, the attp-int loci are poorly conserved. here we demonstrate that phibt1 integrates into a different attachment site than phic31. phibt1 attb lies within sco4848 encoding a 79-amino-acid putative integral membrane protein. integration vectors based on phibt1 integrase were shown to have a broad host range and are fully compatible with those based on the phic31 attp-int locus.200312923110
site-specific recombination in schizosaccharomyces pombe and systematic assembly of a 400kb transgene array in mammalian cells using the integrase of streptomyces phage phibt1.we have established the integrase of the streptomyces phage phibt1 as a tool for eukaryotic genome manipulation. we show that the phibt1 integrase promotes efficient reciprocal and conservative site-specific recombination in vertebrate cells and in schizosaccharomyces pombe, thus establishing the utility of this protein for genome manipulation in a wide range of eukaryotes. we show that the phibt1 integrase can be used in conjunction with cre recombinase to promote the iterative integration of t ...200818096621
dna cleavage is independent of synapsis during streptomyces phage phibt1 integrase-mediated site-specific recombination.bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. integration of ϕbt1 and ϕc31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. both ϕbt1 and ϕc31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕbt1 integrase relaxed supercoiled dna containing a single integration site. systematic mutation of t ...201020871112
site-specific transgene integration in the human genome catalyzed by phibt1 phage integrase.the streptomyces phage phibt1 integrase catalyzes recombination between phage attp and bacterial attb sites (att, attachment), resulting in phage dna integration into the bacterial host genome in a unidirectional manner. multiple pseudo-attb and -attp sites are present serendipitously in mammalian genomes and can recombine with wild-type attp and attb sequences. the phibt1 system has been used previously to achieve site-specific integration of murine phenylalanine hydroxylase cdna into hepatocyt ...200818067406
highly efficient in vitro site-specific recombination system based on streptomyces phage phibt1 integrase.the streptomyces phage phibt1 encodes a site-specific integrase of the large serine recombinase subfamily. in this report, the enzymatic activity of the phibt1 integrase was characterized in vitro. we showed that this integrase has efficient integration activity with substrate dnas containing attb and attp sites, independent of dna supercoiling or cofactors. both intra- and intermolecular recombinations proceed with rapid kinetics. the recombination is highly specific, and no reactions are obser ...200818689469
tandem assembly of the epothilone biosynthetic gene cluster by in vitro site-specific recombination.we describe a site-specific recombination-based tandem assembly (ssrta) method for reconstruction of biological parts in synthetic biology. the system was catalyzed by streptomyces phage φbt1 integrase, which belongs to the large serine recombinase subfamily. this one-step approach was efficient and accurate, and able to join multiple dna molecules in vitro in a defined order. thus, it could have applications in constructing metabolic pathways and genetic networks.201122355658
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