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URL: http://www.addgene.org/33030
Proper Citation: RRID:Addgene_33030
Insert Name: Toca-1
Organism: Homo sapiens
Bacterial Resistance: Ampicillin
Defining Citation: PMID:15260990
Vector Backbone Description: Backbone Size:4350; Vector Backbone:pCS2+MT; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: Molecular cloning of Toca-1 was difficult, because the cDNA encoding human Toca-1 is lethal to E. coli. The toxicity problem was not resolved by using vectors with low copy numbers or with minimal transcriptional activity, suggesting that the source of toxicity was not the translated protein product but rather the DNA sequence itself. The toxic region of the Toca-1 cDNA was localized to codon 127. The Xenopus tropicalis homolog of Toca-1 is completely nontoxic when cloned using E. coli and is identical in amino acid sequence to human Toca-1 between amino acids 124 and 127, but these amino acids are encoded by different codons. Replacing codons 126 and 127 in the human Toca-1 with the X. Tropicalis sequence (which constitute silent mutations) completely ameliorated the toxicity problem and allowed us to propagate the human Toca-1 clone in E. coli without difficulty.
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Source: Addgene
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