Part:BBa_K3128008:Design

Engineered mutant of red fluorescent protein with RFC21 restriction sites

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  Illegal BamHI site found at 685
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 561
  Illegal AgeI site found at 673
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

TAATAA double stop codon added (DE).

Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A. Two restriction sites were added on both sides of RFP gene as such : BglII_RFP_BamHI

From Campbell: The final clone, designated mRFP1, contains a total of 33 mutations relative to DsRed of which 13 are internal to the β-barrel (N42Q, V44A, V71A, K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T, S197I, and T217A). Of the 20 remaining external mutations, three are the aggregation-reducing mutations from T1 (R2A, K5E, and N6D), three are AB interface mutations (I125R, V127T, and I180T), ten are AC interface mutations (R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T, F224G, and L225A), and four are additional beneficial mutations (T21S, H41T, C117E, and V156A).

Source

[http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=nucleotide&dopt=GenBank&list_uids=21464837 Genbank accession AF506027]

1. Campbell pmid=12060735

[http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12060735 URL]

References

<biblio>

1. Zhang pmid=12461557

</biblio>