Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
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Cites methods from "Efficient four fragment cloning for..."
...Both DNA fragments and the predigested pRFHU2 binary vector (Frandsen et al., 2008) were mixed together and treated with the USER enzyme (New England Biolabs) to obtain the deletion vector....
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16 citations
Cites methods from "Efficient four fragment cloning for..."
...The construction of vectors for over-expression and knockout of selective NRPS genes was performed using the well-established USER friendly cloning technique [12]....
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...The transformation vectors were constructed by USER friendly cloning [12], and second generation Illumina and third generation MinION Nanopore sequencing verified that the resulting mutants were correct (Figure S2)....
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...The transformation vectors were constructed by USER friendly cloning [12], and second generation Illumina and third generation MinION Nanopore sequencing verifi d that the r sulting mutants were correct (Figur S2)....
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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
"Efficient four fragment cloning for..." refers background in this paper
...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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