Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
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...To generate a VdSge1 complementation construct, a 2,699-bp EcoRI/PacI fragment containing the VdSge1 coding sequence with a sequence 1,100 bp upstream and 66 bp downstream was amplified from V. dahliae strain JR2 genomic DNA, and cloned into binary vector pBT081 (Houterman et al 2008)....
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Cites methods from "Efficient four fragment cloning for..."
...PCR products were subsequently cloned into pRF-HU2 (Frandsen et al., 2008)....
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Cites methods from "Efficient four fragment cloning for..."
...1A, were obtained by a USER Friendly cloning system (Frandsen et al., 2008)....
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...5 kb upstream and downstream fragments from the promoter and terminator regions were cloned into the plasmid vector pRF-HU2 (Frandsen et al., 2008), a binary vector designed to be used with the USER friendly cloning technique (New England Biolabs, USA), as described previously (Crespo-Sempere et al....
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...…replacement plasmids, 1.5 kb upstream and downstream fragments from the promoter and terminator regions were cloned into the plasmid vector pRF-HU2 (Frandsen et al., 2008), a binary vector designed to be used with the USER friendly cloning technique (New England Biolabs, USA), as described…...
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References
2,471 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Several laboratories have solved this problem by dividing the replacement constructs into two, a technique known as bipartite gene-targeting or split-marker recombination [6- 8]. In this technique, the two HRS's are fused with two thirds of either the 3' or 5'end of the selection marker gene, by fusion-PCR [ 9 ]....
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1,488 citations
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...The use of Proofreading DNA polymerase is essential when making targeted genome modifications in fungi, due to the close spacing of fungal genes [26], which often means that the HRS extends into neighbouring genes or their regulatory sequences....
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1,185 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Examples are the Xi-cloning, InFusion cloning, Ligase independent cloning (LIC-PCR), Recombinational cloning and USER Friendly cloning techniques [16-20]....
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1,074 citations
"Efficient four fragment cloning for..." refers background or methods in this paper
...crassa, Colot and coworks [1], also allows for efficient four fragment cloning....
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...Contrary to Saccharomyces cerevisiae, where 30 bp is sufficient, many filamentous fungi require longer HRS [1], eg Fusarium graminearum needs 400 bp [2] 1500 bp is reported for Aspergillus niger [3] and around 1000 bp for Neurospora crassa [4]....
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...Vector construction for targeted replacement of genes is reduced to design of two primer pairs, which will permit automation of the experimental design as required for high-throughput knockout projects [1]....
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...Recombinational cloning of the two required HRS with a selection marker gene and a vector backbone is carried out in yeast, followed by PCR amplification of the two HRS and selection marker gene [1]....
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893 citations
"Efficient four fragment cloning for..." refers background in this paper
...The Agrobacterium tumefaciens mediated transformation (ATMT) technology [10] has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types [11]....
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