Part:BBa_K243000:Design

Protein domain (active) of the restriction endonuclease FokI

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 487
  

Design Notes

Planning the design of two different FokI-heterodimers

• extract coding region of Fok from the restriction-modification genes of the chromosomal DNA of Flavobacterium okeanokoites [http://www.ncbi.nlm.nih.gov/nuccore/148723?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum F.okeanokoites fokIR and fokIM genes]
  
• delete the first 1158 nucleotides/386 aa (recognition domain)
  
• switch Cystein 541/463-465 to Ser (TGT->TCT)
  
  

Modifications of the single vectors to introduce heterodimeric modifications according to Miller_J,_Rebar_E_Nature_biotech_2007_25_778

Modifications of the first vector (catalytic active heterodimer)
-heterodimeric aminio acids

• switch Glutamate 490/310-312 to Lysin (GAA->AAA)
  
• switch isoleucin 538/454-456 to Lysin (ATC->AAA)
  

Modifications of the second vector (catalytic inactive heterodimer)
-heterodimeric amino acids

• switch Glutamin 486/298-300 to Glutamate (CAA->GAA)
  
• switch Isoleucin 499/337-339 to Leucin (ATC->CTG)
  

-catalytic amino acids

• switch Aspartate 450/190-192 to Alanin (GAC->GCG)
  
• switch Aspartate 467/243-245 to Alanin (GAT->GCG)
  

Annotations:

• The notation e.g. for Cystein 541/463-465 means the amino acid 541 in literature which correspond to the codons 463-465 in our vector.
  
• For exchanging the amino acids we used the Codon usage table in E.coli from Hénaut and Danchin. [http://www.faculty.ucr.edu/~mmaduro/codonusage/codontable.htm E.coli Codon Usage]
  
  

Source

extract coding region of Fok from the restriction-modification genes of the chromosomal DNA of Flavobacterium okeanokoites. Part synthesized by Mr.Gene

References

Mary C. Looneya, Laurie S. Morana, William E. Jacka, George R. Feeherya, Jack S. Bennera, Barton E. Slatkoa and Geoffrey G. Wilson;(1989)
Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase; Gene Vol.80 Issue:2 Pages:193-208

Jeffrey C Miller1, Michael C Holmes1, Jianbin Wang1, Dmitry Y Guschin1, Ya-Li Lee1, Igor Rupniewski1, Christian M Beausejour1,2, Adam J Waite1, Nathaniel S Wang1, Kenneth A Kim1, Philip D Gregory1, Carl O Pabo1,2 & Edward J Rebar (2007);
An improved zinc-finger nuclease architecture for highly specific genome editing; Nature Biotechnology 25, 778 - 785