StarGate Combinatorial Cloning Systems
StarGate Cloning Technology
Easy combinatorial cloning in one tube
- 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 StarGate Combi Entry Cloning Set containing all required components to subclone and (if desired) mutate 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.
Easy combinatorial cloning in one tube
The StarGate Technology
In a first step, the gene of interest (GOI) is equipped at both ends with combinatorial sites (4 bases) by PCR and is inserted into an Entry Vector by a simple one-tube reaction. The Entry Vector is provided in linearized form. Since religation of the vector is virtually excluded, it reacts efficiently with a blunt end PCR product comprising GOI with flanking combinatorial sites. After sequence confirmation, the resulting Donor Vector is the origin for the highly parallel subcloning of GOI into a multitude of Acceptor Vectors, each providing a different genetic surrounding like host specific promoters and different purification tags by a second simple one-tube reaction. Thus, the GOI may be transferred in a second step from the Donor Vector into various Acceptor Vectors providing the desired genetic surrounding (i.e. tag, promoter, signal sequence etc.) by means of StarCombinase to create corresponding Destination Vectors. The desired E. coli clones carrying a Destination Vector can easily be identified through blue/white selection. The resulting Destination Vectors are then transformed into the corresponding host cells for further experiments. The Destination Vector of the left example places the GOI under control of the CMV promoter allowing GOI expression in mammalian cells. In addition, a tag is fused to the C-terminal end of the GOI expression product.
The TAGnology
StarGate is a technology that allows the systematic combination of promoters (i.e. host-specific promoters such as Tet, T7, CMV, CUP1, polyhedrin), purification tag sequences (e.g. 6xHis-tag, Strep-tag, GST, FLAG) or other genetic elements with any gene of interest (GOI) in a convenient cloning system resulting in optimal gene expression in a chosen host system (E. coli, yeast, mammalian or insect cells).
The core element of this new technology is the site-specific combinatorial enzyme StarCombinase™, that makes cloning versatile, fast, easy and safe. This enzyme is included (as part of the StarSolution) in the StarGate Combi Entry Cloning Set as well as in the StarGate Transfer Reagent Set. Our easy-to-use software StarPrimer D'Signer facilitates the design of primers for the ENTRY Vectors both for StarGate Standard Entry Cloning and for StarGate Mutagenesis Cloning. The software guides you step-by-step through the whole PCR procedure.
For getting started with StarGate, you need two Sets only:
For StarGate Standard Cloning, you use the StarGate Combi Entry Cloning Set to create up to 20 different Donor Vectors containing your genes of interest. The new StarGate Entry Cloning Set provides the combination of Standard and Mutagenesis Entry Cloning in one set and an improved Entry Cloning Step.
After having created your Donor Vector(s), you use the StarGate Transfer Reagent Set (20 reactions) to transfer each gene of interest from the Donor Vector directly into different Acceptor Vectors you can choose from our wide range of sophisticated Acceptor Vectors specifically designed for your requirements. The final cloning product is your Destination Vector that can be used for transformation/transfection of your host cells (E. coli, yeast, insect or mammalian cells).
Please download list with available Acceptor Vectors and select the vectors you require
The StarGate Combi Entry Cloning Set (20 reactions) includes the Entry Vector pENTRY-IBA51, StarSolutions M1 - M3, For/Rev Seqencing Primers, competent E. coli Top10 cells (for 20 transformations) as well as a DNA Ladder (DNA Ruler).
The Transfer Reagent Set you require to transfer your target gene from the Donor Vector to the desired Acceptor Vector consists of the three Star Solutions A1 - A3 (20 transfer reactions).
The StarGate Newcomer Entry Set is a very cost-effective offer for scientists starting to work with StarGate. It includes the StarGate Combi Entry Cloning Set as well as the Transfer Reagent Set and Control Reactions (PCR fragment and Donor Vector). The Newcomer Set provides all products required to perform the entire StarGate procedure to a significantly reduced price compared to single components.
Save up to 45% when buying the StarGate Newcomer Set!
StarGate Mutagenesis System
Convenient tool for site-directed mutagenesis
- Construction of Donor Vector with mutated GOI (please click to enlarge).
- Fully compatible with StarGate system
- Uncomplicated generation of Donor mutants
- Parallel automated mutagenesis possible
- Attractive prices
The StarGate Combi Entry Cloning Set also provides 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 (with wild-type GOIs) 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 free software StarPrimer D'Signer. 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 Cloning, you use the StarGate Combi Entry Cloning Set to create up to 20 different Donor Vectors containing your mutated genes of interest. Thereafter, you use the StarGate Transfer Reagent Set as described above.
The StarGate Combi Entry Cloning Set (20 reactions) includes the Entry Vector pENTRY-IBA51, StarSolution M1, M2 & M3, For/Rev Seqencing Primers, competent E. coli Top10 cells (for 20 transformations) as well as a DNA Ladder (DNA Ruler).
StarGate Fusion Cloning System
For generating fusion proteins and constructing artificial operons
- Principle of StarGate Fusion Cloning (please click to enlarge).
- 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 - combine your genes in any (variable) order
- 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-IBA11) 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 pNFUSE-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-IBA51; 5 rxns), Downstream Fusion Vector pCFUSE-IBA11 (5 rxns), StarSolutions F1 to F6 (for 5 fusions), Upstream Fusion Vector pNFUSE-IBA-IRES11 (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-IBA51; 5 rxns), Downstream Fusion Vector pCFUSE-IBA11 (5 rxns), StarSolutions F1 to F6 (for 5 fusions), Upstream Fusion Vector pNFUSE-IBA-SD11 (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-IBA51; 5 rxns), Downstream Fusion Vector pCFUSE-IBA11 (5 rxns), StarSolutions F1 to F6 (for 5 fusions), Upstream Fusion Vector pNFUSE-IBA-LINK11 (5 rxns), competent E. coli TOP10 cells (20 transformations).
Order Information
Note: Acceptor Vectors used with the StarGate Transfer Reagent Set are not included in the StarGate kits and have to be ordered separately. They are also not provided with StarGate Transfer Reagent Set. Please choose type and number of vectors from the Acceptor Vector list.
Save up to 40%* and get 2 Acceptor Vectors for free when ordering the StarGate Combi Newcomer Set!
*compared to buying the individual StarGate products
The StarGate Technolgy is distributed by PromoCell for IBA GmbH, Germany.