User guide:

  • "File upload" : Please upload a fasta file containing only the open reading frame to be analyzed. Only DNA base characters are allowed (G, A, T, C). Note that the first amber stop codon context to be analyzed by iPASS is at the third codon of the open reading frame (nucleotide context: -6 to -1 and +4 to +9, with TAG at +1, +2, +3).
  • "Sequence input" : Please paste the open reading frame to be analyzed and use the "Submit" button to upload your sequence. Only DNA base characters are allowed (G, A, T, C). Note that the first amber stop codon context to be analyzed by iPASS is at the third codon of the open reading frame (nucleotide context: -6 to -1 and +4 to +9, with TAG at +1, +2, +3).
  • "Optimize context" : Check this box to optimize the iPASS score and hence ncAA incorporation efficiency by synonymous exchange of codons flanking the amber stop codon.

Output:

An iPASS score of ≥ 1 should indicate above average relative ncAA incorporation efficiency. A minimal iPASS score difference of ca. 2.5 after amber stop codon context optimization usually improves ncAA incorporation efficiency. Please note that the iPASS tool has been developed to predict and optimize the suppression of amber stop codons in mammalian cells using the orthogonal Methanosarcina mazei pyrrolysyl-tRNA synthetase/tRNA Pyl CUA pair. Hence, iPASS might not reliably predict ncAA incorporation efficiencies for other genetic code expansion strategies (e.g. tyrosyl amber suppressor tRNAs) or eukaryotic organisms (e.g. yeast).

Error report and feedback:

Please report errors and bugs to bultmann(at)bio.lmu.de. We would also appreciate to receive feedback on how well the tool predicts relative ncAA incorporation efficiencies in your applications and cell lines.

How to cite:

The iPASS tool was developed by your colleagues. Please cite the publication in which iPASS has been described in your Material and Methods section: Bartoschek MD, Ugur E, Nguyen TA, Rodschinka G, Wierer M, Lang K, Bultmann S. Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells (2021).

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