Part:BBa_K5142034

Azidophenylalanine-specific tyrosyl-tRNA synthetase

This part includes the coding sequence (cds) of an azidophenylalanine-specific variant of E. coli tyrosyl-tRNA synthetase (pAzpaRS). pAzpaRS can transfer 4-azido-L-phenylalanine (4AzF) to the amber suppressor tRNA, forming the corresponding aminoacyl-tRNA. We used pAzpaRS to synthesize the amber suppressor tRNA carrying 4AzF, which was used to translate the amber suppressor codons in A27L-3stop gene, thereby inserting 4AzF into the mutant A27L protein.

This part was subcloned from the plasmid pcpAzpaRS_v2 (Addgene Plasmid #196485) (1), which was originally derived from the TyrRS gene of E.coli.
Reference

1. Hino N, Sakamoto K, Yokoyama S. Site-specific incorporation of unnatural amino acids into proteins in mammalian cells. Methods Mol Biol. 2012;794:215-28.

pAzpaRS was designed to specifically aminoacylate the amber suppressor tRNA (see BBa_K5142008) without cross-reacting with endogenous tRNAs.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 1293
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal SpeI site found at 1294
  Illegal PstI site found at 1308
  Illegal NotI site found at 7
  Illegal NotI site found at 1301
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal BamHI site found at 758
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 1294
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal XbaI site found at 16
  Illegal SpeI site found at 1294
  Illegal PstI site found at 1308
  
• 1000
  COMPATIBLE WITH RFC[1000]