pCS2+MT-hToca-1-W518K
(Plasmid #33031)

• Depositing Lab: Marc Kirschner
• Publication: Ho et al Cell. 2004 Jul 23;118(2):203-16.

Backbone

• Vector backbone: pCS2+MT
• Backbone size w/o insert (bp): 4350
• Vector type: Mammalian Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Toca-1
• Species: H. sapiens (human)
• Mutation: W518K
• Entrez Gene: FNBP1L (a.k.a. C1orf39, TOCA1)
• Promoter: simian CMV IE94
• Tag / Fusion Protein:
  • 6xMyc (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: none (unknown if destroyed)
• 3′ cloning site: none (unknown if destroyed)
• 5′ sequencing primer: SP6
• 3′ sequencing primer: M13 reverse

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.