USER cloning


USER cloning



The USER Friendly technology

The Uracil-Specific Excision Reagent (short USER) FriendlyTM cloning technology is dependent on the excision of a single uracil base from either end of a PCR generated insert. The uracil bases are included in the primers as part of a 9 bp long 5’ extension. Following PCR amplification with the primers, excision of the uracil bases results in the generation of 9 bp long 3’ overhangs on the PCR product complementary to 3’ overhangs in the vector fragments. The 3’ overhangs in the vector DNA is generated by the combined cutting of PacI (standard restriction enzyme) and Nt.Bbc.CI (nicking enzyme).

Annealing of the 9 bp long sequences in the PCR insert and the vector DNA is stable enough to survive chemical transformation into E. coli. Inside E. coli the DNA fragments are covalently linked by the formation of phosphodiester bonds between the free DNA ends.



Figure:          Top) Vector with the two new unique USER cloning sites, that each consists of a PacI site, two Nt.BbvC IB sites and two times two variable bases. The variable bases, shown in yellow, green, gray and pink boxes, ensure directional cloning of the PCR amplified inserts. Bottom) The two vector fragments following the combined cutting of PacI and Nt.BbvC IB and purification. PacI is a standard restriction enzyme that cuts both strands, while Nt.BbvC IB is a nicking enzyme that only cuts one strand.


Instruction for use of the USER Friendly cloning system for targeted gene replacement in fungi 




To obtain the Agrobacterium tumefaciens plasmid with the shown USER Friendly cloning sites contact:

          Rasmus Frandsen by e-mail (ras(at)


          The Fungal Genetics Stock Center (




Articles dealing with the USER Friendly cloning technique:

bullet Rasmus J.N. Frandsen, Jens A. Andersson, Matilde B. Kristensen and Henriette Giese: "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi", BMC Molecular Biology 2008, 9:70. OPEN ACCESS
bulletNour-Eldin HH, Hansen BG, Nørholm MH, Jensen JK, Halkier BA: "Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments", Nucleic Acids Res. 2006;34(18):e122. Epub 2006
bulletBitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R.: "USER friendly DNA engineering and cloning method by uracil excision", Nucleic Acids Res. 2007;35(6):1992-2002.
bulletFernando Geu-Flores, Hussam H. Nour-Eldin, Morten T. Nielsen, and Barbara A. Halkier: "USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products", Nucleic Acids Res. 2007;35(7):e55. Epub 2007






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Dette sted blev sidst opdateret 10. July 2010