Fusion Cloning

Generate fusion proteins or construct artificial operons for multiple gene expression in bacterial or mammalian cells

Fuse genes from any Donor Vector in any combination to make single fusion proteins or even to construct artificial operons for multiple gene expression in bacterial or mammalian cells. The StarGate¢ç Fusion Cloning system allows easy and fast fusion of two different genes of interest (GOI-1 and GOI-2) by an intergenic region (IR). Only two sequential StarGate¢ç subcloning reactions with dedicated Fusion Vectors are required. The procedure can be repeated to fuse more than two GOI¢¥s.

Fusion Cloning Procedure

StarGate¢ç fusion cloning is an optional intermediate step between the Entry reaction and the Transfer reaction. It allows easy and fast fusion of two genes of interest (GOI-1 and GOI-2) present in separate Donor Vectors by performing two sequential StarGate¢ç subcloning reactions.
 
StarGate_FusionCloning_detailsISv3-gr-997ca529.png
The first subcloning reaction is placing each GOI into a special Fusion Vector. One Fusion Vector (pNFUSE-IBA-derivative specifying the intergenic region) is designed for upstream, while the other (pCFUSE-IBA) is for downstream positioning of the respectively inserted GOI. The Downstream Fusion Vector is identical in each reaction. Upstream and downstream Fusion Vectors with inserted GOI are then assembled in a directed manner within pENTRY-IBA51 thereby providing a new Donor Vector consisting of GOI1 fused to GOI2 by the intergenic region of the given pcFUSE-IBA derivative.
The fused GOIs are now ready to be subcloned into any of the StarGate¢ç
Acceptor Vectors available for expression ("transfer reaction"). Upstream Fusion Vectors with different intergenic regions are included as part of our new Fusion Cloning Sets. 
 
StarGate¢ç Fusion Cloning Options
 
 
stargate_newsletterIssue11S5-5e481316.png
The StarGate¢ç Fusion Cloning system allows easy and fast fusion of two different genes of interest (GOI-1 and GOI-2) by an intergenic region (IR). Such an intergenic region may e.g. code for an amino acid linker sequence directly connecting, after performing the fusion procedure, the gene product of a first GOI to the product of a second GOI (LINK). Alternatively, it may include a Shine Dalgarno (SD) or an IRES site for the construction of synthetic operons in bacterial or mammalian cells, respectively.

StarGate¢ç Fusion Cloning Sets

Three different versions of StarGate¢ç Fusion Cloning Sets are available to date. Please note, that also a combination of these three versions is possible allowing the construction of a large variety of genetic fusions. Your gene of interest could e.g. be fused with GFP using "LINK1" followed by "SD1" to construct an artificial operon of your GFP fusion reporter protein with further genes. For this purpose, the Upstream Fusion Vectors are also available separately on request.
For detailed informations on the „intergenic regions (IR)¡± have a look on technical informations.

 
 

StarGate¢ç Fusion Cloning Sets and respective intergenic regions

  • StarGate¢ç Fusion Cloning Set "IRES1"
    Internal ribosomal entry site (IRES) for polycistronic gene expression in mammalian cells from one expression vector
     
  • StarGate¢ç Fusion Cloning Set "SD1"
    Shine-Dalgarno (SD) sequences for the construction of artificial operons in
    E. coli
     
  • StarGate¢ç Fusion Cloning Set "LINK1"
    Amino acid linker (GSGGGSGGGS) for the generation of fusion proteins
     
  • StarGate¢ç Fusion Cloning Set „LINK2¡°
Amino acid linker (GlySerGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) for the generation of fusion proteins
 

StarGate¢ç intergenic region cassettes

 

Available Sequencing primers for Fusion Vectors

description Amount cat. no.
Forward sequencing primer for Fusion vectors
(pCFUSE/pNFUSE); HPLC-purified
1 nmol 5-0000-155
Reverse sequencing primer for Fusion vectors
(pCFUSE/pNFUSE); HPLC-purified
1 nmol 5-0000-156
Forw. and rev. seq. primers for Fusion vectors
(pCFUSE/pNFUSE); HPLC-purified
1 nmol
each
5-0000-157

References

  1. Belfield, EJ, Hughes RK, Tsesmetzis, N, Naldrett, MJ, Casey, R (2007). 
    The gateway pDEST17 expression vector encodes a –1 ribosomal frameshifting sequence.
    NAR 35 (4), 1322-1332.
  2. Boshart, M, Weber, F, Jahn, G, Dorsch-Häsler, K, Fleckenstein, B, and Schaffner, W (1985). 
    A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus.
    Cell 41, 521-530.
  3. Gietz, RD, Sugino, A (1988). 
    New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527-34. Macreadie, IG, Horaitis, O, Verkuylen, AJ, Savin, KW (1991). Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast
    Gene 104, 107-11.
  4. Nelson, JA, Reynolds-Kohler, C, and Smith, BA (1987). 
    Negative and Positive Regulation by a Short Segment in the 5¢¥-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene. 
    Mol. Cell. Biol. 7, 4125-4129.
  5. Skerra, A. (1994). 
    Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli.
    Gene 151, 131-135.
  6. Studier, FW, Rosenberg, AH, Dunn, JJ, Dubendorff, JW (1990). 
    Use of the T7 RNA polymerase to direct expression of cloned genes.
    Meth. Enzymol. 185, 60-89.
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