EENdb - Utilities  >  ZFN framework & linker peptide
Summary
The framework/scaffold of ZFN contains the sequences beside the variable 7-aa regions of each finger. Not all frameworks are reported by original refereces. ZFNs constructed by OPEN, CoDA and "PRS" often use the framework/scaffold from ZIF168. ZFNs constructed by MA use multiple sources of single- or double-finger modules, which may be from various frameworks. Other C2H2 frameworks besides ZIF268 are also used, with potential modification.

The linker peptide of ZFN is defined as the amino acid sequence between the last histidine (H) of ZFP DNA-binding domain and the beginning of FokI cleavage domain (QLVKS...). Different lengths of linker peptide have been reported to prefer different length of spacer.

Sequence of a typical ZFN with framework ZIF268
Sequence (posterior region of FokI cleavage domain omitted), based on aa 334-416 of mouse ZIF268/EGR1

M A P K K K R K V D Y K D H D G D Y K D H D I D Y K D D D D K G T R P Y A C P V E S C D R R F S R S D E L T R H I R I H T G Q K P F Q C R I C M R N F S R S D H L T T H I R T H T G E K P F A C D I C G R K F A R S D E R K R H T K I H L R G S Q L K V S ...


Sequence with annotation:

|< SV40 NLS>| |<---------- Tag (e.g. 3xFlag) ---------->| GT: Tag-ZFP linker peptide (L.) M A P K K K R K V D Y K D H D G D Y K D H D I D Y K D D D D K G T |<--------------------- Fn ---------------------->| (*: key aa of "C2H2") * * |<- F1 7aa->| * * TG: F1-F2 L. (other F-F L. also used) R P Y A C P V E S C D R R F S R S D E L T R H I R I H T G * * |<- F2 7aa->| * * TG: F2-F3 L. Q K P F Q C R I C M R N F S R S D H L T T H I R T H T G * * |<- F3 7aa->| * * LRGS: one type of ZFP-FokI L. E K P F A C D I C G R K F A R S D E R K R H T K I H L R G S QLKVS...: FokI cleavage domain Q L K V S ...


Types of ZFP-FokI linker peptide
LinkerLengthPreferred SpacerCommon UsageComments
TG(G4S)3 17 aa (variable) Early works of ZFN targeting artificial sequences Usually called "(G4S)3" linker
QNKK 4 aa 6 nt Early works of ZFN targeting artificial sequences Usually called "no-linker"
LRGS 4 aa 5-6 nt ZFNs constructed by OPEN, CoDA or Sangamo
TGEK 4 aa 5-6 nt ZFNs constructed by MA from ToolGen Inc.
TGGS 4 aa 5-6 nt ZFNs constructed by MA or PRS
(more) Other works



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