Gateway Cloning Service

The Gateway Cloning System is a highly efficient, high-throughput molecular biology platform developed by Invitrogen (now Thermo Fisher). It allows researchers to seamlessly transfer a gene of interest (GoI) from a universal Entry Clone into multiple, functionally diverse Destination Vectors without using restriction enzymes or ligase. This site-specific recombination technology enables rapid screening of expression systems in various host organisms (e.g., bacterial, yeast, mammalian, plant) and under different regulatory controls (e.g., inducible, constitutive).

CD Biosynsis offers a comprehensive Gateway Cloning Service designed to accelerate your functional studies. We handle the entire process, from synthesizing your GoI and constructing the initial Entry Clone (attL flanked) to performing the final LR and BP recombination reactions into any standard or custom Destination Vector. Our automated, high-fidelity platform guarantees successful production of sequence-verified Expression Clones for rapid and parallel functional analysis.

Highlights

Key advantages of using our high-throughput Gateway Cloning platform:

• Restriction-Free Transfer: Eliminates the tedious steps of restriction digest, gel purification, and ligation, saving significant time and reducing potential errors.
• High Success Rate: Guaranteed transfer efficiency (BP or LR reaction) with highly efficient recombinase mixes and positive selection markers.
• Universal Compatibility: Compatible with hundreds of commercial Gateway-adapted Destination Vectors for expression in virtually any host system.
• High-Throughput Format: Services are optimized for high-throughput parallel cloning, allowing simultaneous creation of numerous Expression Clones from one Entry Clone or multiple Entry Clones.

Applications

Gateway Cloning is essential for large-scale, functional genomics projects and protein expression screening:

Protein Expression Screening

           

Rapidly transferring a GoI into multiple Destination Vectors (e.g., with different tags or promoters) to find the optimal expression system.

Functional Genomics & cDNA Libraries

High-throughput cloning of entire cDNA libraries into an Entry Clone collection for eventual rapid transfer to functional assay vectors.

Dual-Hybridization Systems

Efficiently cloning interacting proteins into Destination Vectors for Yeast Two-Hybrid or mammalian Protein-Protein Interaction studies.

Protein Tagging & Localization

Seamlessly fusing GoIs with various N or C-terminal GFP, His-tag, Flag-tag or purification tags using compatible Destination Vectors.

Products & Workflow Options

We provide flexible options to enter the Gateway workflow at any stage:

Option 1: Entry Clone Construction (BP Reaction)

We synthesize your GoI and clone it into a pDONR Vector (attP x attB -> attL flanked Entry Clone). Ideal starting point.

Option 2: Expression Clone Generation (LR Reaction)

Using a client-provided or internal Entry Clone, we perform the LR reaction into your specified Destination Vector (attL x attR -> attB).

Option 3: Multi-Fragment Assembly (MultiSite)

Cloning of two or more Entry Clones (e.g., Promoter, GoI, Tag) simultaneously into a MultiSite Destination Vector.

Destination Vector Customization

We can adapt your existing proprietary vector to be Gateway-compatible by inserting the necessary attR recombination sites and ccdB selection cassette.

High-Throughput Cloning Arrays

Automated parallel LR reactions for transferring dozens to hundreds of Entry Clones into one or more Destination Vectors in 96-well format.

Workflow

Our integrated workflow ensures high-yield, sequence-verified Gateway clones:

• Project Design and GoI Preparation: You specify your gene(s) and the target Destination Vector(s). We optimize codon usage, synthesize the GoI, and add the required attB sites.
• BP Reaction (Entry Clone Construction): The attB-flanked GoI is recombined into the pDONR Vector (attP x attB) using BP Clonase to create the attL-flanked Entry Clone.
• LR Reaction (Expression Clone Generation): The Entry Clone (attL) is recombined with the Destination Vector (attR) using LR Clonase to create the final Expression Clone (attB).
• Selection and Quality Control: The reaction is transformed into E. coli (positive selection against ccdB gene). All final Expression Clones are Sanger sequence verified across the recombination sites and the entire GoI.
• Delivery: Delivery includes purified plasmid DNA and bacterial glycerol stock for the final Expression Clone(s), along with COA and Sanger sequencing data.

We provide the assurance needed for multi-vector expression screening:

• Guaranteed Fidelity: Gateway is an extremely high-fidelity process; we guarantee the integrity of your sequence throughout the recombination and cloning steps.
• Time Savings: The parallel nature of Gateway allows for simultaneous cloning into dozens of vectors, dramatically reducing project timelines for expression optimization.
• Expertise in MultiSite: Specialized handling of MultiSite Gateway assembly to correctly orient and clone up to four fragments in a single reaction.
• ccdB Vector Handling: Specialized host strains for propagating Destination Vectors containing the ccdB gene, ensuring template integrity.