• Convenient subcloning of your gene of interest into diverse high-level expression vectors with different properties and for different host systems (e.g. mammalian, yeast, E. coli, baculovirus)
• Ready-to-use kits containing all required components to subclone your gene of interest
• Minimal modification of the gene of interest due to extremely short combinatorial sites (4 bases only)
• Improved, systematic screening of different elements (e.g. different tags/promoters) and different host/tag combinations in a variety of hosts
• Inherent highest level cloning efficiency due to a directed reaction (no equilibrium)
• Availability of a multitude of combinatorial sites for combinatorial cloning
• One-tube, easy-to-handle subcloning procedure (1 hour)
• Cutting bands out of gels becomes superfluous  
• Acceptor Vectors for different hosts with correlating features always yield exactly the same protein sequence  
• Once Donor Vector is sequenced no further verification is required
• Polycistronic gene expression readily accessible
• Easy generation of fusion proteins
• Versatile mutagenesis system with StarPrimerD'Signer software
• Cost-effective StarGate products

Efficient procedures to generate functional recombinant proteins or protein complexes are of key importance in state-of-the-art life sciences. Many tools like various expression hosts (bacteria, yeast, insect and mammalian cells), promoters, affinity or fluorescent tags are currently available to express, purify, detect or immobilize recombinant proteins. Due to the diverse nature of proteins, however, it is impossible to predict which combination of these tools will perform best. Therefore, many have to be tried in order to identify the optimal solution.

To systemize and accelerate this initial search which is crucial for successful subsequent proteomic research, we now provide the StarGate Cloning System. StarGate offers rapid and highly efficient subcloning of an arbitrary gene - initially cloned into a Donor Vector - to simultaneously fuse it with many different genetic surroundings via transfer into Acceptor Vectors to generate Destination Vectors. The latter enable the efficient expression of your protein with various features (e.g. different tags and different promoters) in different hosts. 

StarGate Principle (animation)

StarGate Transfer Reaction Mechanism (animation)

• Fully compatible with StarGate system
• Uncomplicated generation of Donor mutants
• Parallel automated mutagenesis possible
• Attractive prices

The StarGate Mutagenesis Entry Cloning Set is a convenient tool for site-specific modification of the gene of interest (GOI) - within a sequence stretch of up to 80 bases in length - during generation of the Donor Vector. The only difference to the standard StarGate cloning procedure is that two PCRs have to be performed instead of a single PCR whereby the gene internal PCR primers introduce the desired nucleic acid sequence changes. The design of the essential primers is performed using the easy-to-use software StarPrimer D'Signer coming with the set. To design the primers the sequence of the gene of interest (GOI) as present in the template as well as the desired mutated gene sequence simply have to be pasted into the respective StarPrimer D'Signer sequence windows. The software displays the sequence of the desired primers. Once PCR has been performed using the StarPrimer D'Signer developed primers, both PCR products are mixed with pENTRY-IBA and StarCombinase and are incubated for one hour. After transformation, E. coli clones harbouring the desired Donor clone with the mutated GOI can be easily detected using blue/white selection.
The StarPrimer D'Signer does not only help with the primer design, it will also guide you step by step through the whole PCR process.

For StarGate Mutagenesis Entry Cloning, you use the StarGate Mutagenesis Entry Cloning Set to create up to 5 different Donor Vectors containing your mutated genes of interest. Thereafter, you use the StarGate Transfer Reagent Set as described above.
The StarGate Mutagenesis Entry Cloning Set includes the Entry Vector pENTRY-IBA, StarSolution M1, M2 & M3, For/Rev Seqencing Primers, competent E. coli Top10 cells as well as a DNA Ladder (DNA Ruler) and the StarPrimer D'Signer Software.

• Easy and fast generation of stable fusion proteins
• Polycistronic gene expression in mammalian cell
• Analysis of polycistronic gene expression and gene function
• Versatile combination of multiple target genes of interest
• Protein interaction studies
• Facilitated construction of artificial operons for multiple gene expression in bacterial cells

For several applications, it is interesting to bring two or more genes, already cloned in separate Donor vectors, into operative linkage by an intergenic region. 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. Alternatively, it may include a Shine Dalgarno or an IRES site for the construction of synthetic operons in bacterial or mammalian cells, respectively. StarGate fusion cloning is an optional intermediate step between the StarGate 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 subcloning reactions.

Priniple of StarGate Fusion Cloning:

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-IBA20 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 Acceptor Vectors available for expression ("transfer reaction"). Upstream Fusion Vectors with different intergenic regions are included as part of our new Fusion Cloning Sets.

Three different versions of StarGate 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.

StarGate Fusion Cloning Set IRES1: Internal ribosomal entry site (IRES) for polycistronic gene expression in mammalian cells from one expression vector. The StarGate Fusion Cloning Set IRES1 includes StarGate Fusion Reagent Set (pENTRY-IBA20; 5 rxns), Downstream Fusion Vector pCFUSE-IBA1 (5 rxns), StarSolutions F1 to F6 (for 5 fusions), Upstream Fusion Vector pNFUSE-IBA-IRES1 (5 rxns), competent E. coli TOP10 cells (20 transformations).

StarGate Fusion Cloning Set SD1: It has a Shine-Dalgarno (SD) sequences for the construction of artificial operons in E. coli. The StarGate Fusion Cloning Set SD1 includes StarGate Fusion Reagent Set (pENTRY-IBA20; 5 rxns), Downstream Fusion Vector pCFUSE-IBA1 (5 rxns), StarSolutions F1 to F6 (for 5 fusions), Upstream Fusion Vector pNFUSE-IBA-SD1 (5 rxns), competent E. coli TOP10 cells (20 transformations).

StarGate Fusion Cloning Set LINK1: Provides an amino acid linker (GSGGGSGGGS) for the generation of fusion proteins. The StarGate Fusion Cloning Set LINK1 includes StarGate Fusion Reagent Set (pENTRY-IBA20; 5 rxns), Downstream Fusion Vector pCFUSE-IBA1 (5 rxns), StarSolutions F1 to F6 (for 5 fusions), Upstream Fusion Vector pNFUSE-IBA-LINK1 (5 rxns), competent E. coli TOP10 cells (20 transformations).