View source for Help:Standards/Assembly/RFC25

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==Philosophy==
The Freiburg 2007 iGEM Team designed the Freiburg Standard RFC[25] as an extension of the BioBrick Standard RFC[10], which adds two restriction sites while maintaining the BioBrick sites in the prefix and suffix. The motivation for the Freiburg Standard is to allow for in-frame protein fusions, while also avoiding some of the issues with the Silver Standard. 


===Advantages===
* in-frame fusion of protein parts
* benign protein scar
* N-end rule safe (long protein half-life)
* provision for preserving native N-terminal while using RBS parts
* both new enzymes can be heat-inactivated
* stand-alone protein expression (start + stop in prefix / suffix)
* full BBa compatibility -- functionally & compositionally equivalent to BBa protein coding part
* blunt-cutting isochizomer of NgoMIV (NaeI) -- possibility of directional cloning with two inner restriction sites enables part transfer between different formats and other potentially interesting transfer reactions.

===Disadvantages===
* N-parts are assembled with a different enzyme combination.
* not compatible to BioFusion protein parts (frame shift + stop codon), but see below!


==Technical Specifications==
===Compatability/Illegal Sites===
In order for a part to be compatible with the Freiburg Standard it must not contain the following restriction sites, as these will need to be unique to the prefix and suffix:
*EcoRI site:   GAATTC
*XbaI site: TCTAGA	
*NgoMIV site: GCCGGC	
*AgeI site: ACCGGT	
*SpeI site:    ACTAGT
*PstI site:    CTGCAG
*NotI site:    GCGGCCGC


===Prefix and Suffix===

              Prefix                                     Suffix
 5' - GAATTC GCGGCCGC T TCTAGA TG GCCGGC...part... ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG - 3'
      EcoRI    NotI      XbaI     NgoMIV            AgeI        SpeI     NotI    PstI 

Assembling two parts leaves the following scar:

 5' [part A] ACTAGA [part B] 3'


===Primers===


==Notes?==
===Related Links===
*Standards
*3A Assembly
*Gibson Assembly


===Sources===
*[http://dspace.mit.edu/handle/1721.1/45140 Fusion Protein (Freiburg) Biobrick assembly standard]
*[http://openwetware.org/wiki/The_BioBricks_Foundation:Standards/Technical/Formats#BBF_RFC_25_.28aka_Fusion_parts.2C_Freiburg_iGem2007_team.29 The BioBricks Foundation:Standards/Technical/Formats BioBrick BB-2]
*[[Help:Assembly_standard_25]]


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