Gateway Vector Construction and Cloning Services
A Universal Platform
Gateway™ recombination cloning technology circumvents traditional restriction enzyme–based cloning limitations, providing a universal system for protein expression and analysis based on lambda-phage recombination. Simply clone your gene of interest into a pDONR™ vector to create an “Entry clone”, then recombine the Entry clone with any one of dozens of Gateway-enabled expression vectors to test protein production and function in multiple systems in parallel.
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Why Gateway Cloning?
Widely adopted in the research community with more than 1,500 references since its launch, Gateway cloning is the technology of choice for versatile, high-throughput cloning:
- Fast, 1 hr room-temperature cloning reactions with >99% efficiency deliver the clone you need
- Maintaining orientation and reading frame without using restriction enzymes or ligation makes expression-ready clones
- Eliminating resequencing ensures consistent results throughout your experiment using the same clone from target identification to validation
- Shuttling insert DNA from one expression vector to another affords flexibility while simplifying your cloning workflow
Entry and Expression Clone Services
Supply a full-length cDNA sequence, and our scientists will amplify the open reading frame (ORF; Figure 1) and subsequently clone the PCR product into a Gateway pDONR™ vector to create an Entry clone that is:
- Fully compatible with the Gateway technology platform
- Adaptable to multiple destination vectors
- Ready for protein expression and functional analysis
Once a gene is converted to an Entry clone, it is easily moved into one or more vectors in the desired orientation and reading frame. We can then transfer the ORF in the Entry clone into any Gateway protein expression vector. ORF cloning is particularly valuable if you are conducting gene functional analysis, where the 5´ and 3´ untranslated regions of full-length genes can interfere with optimal protein expression.
|Figure 1. PCR-based ORF cloning: ORFs are amplified using gene-specific primers which contain attB1 and attB2 recombination sites. The resulting amplicon may be recombined with an attP Donor vector (pDONR™) in a B x P reaction to yield an Entry clone. ORFs contained within Entry clones are flanked by attL sites and may be subsequently recombined with any Gateway Destination vector to yield expression clones. Expression clones may, in turn, be used in functional studies.|
High-Throughput ORF Cloning Services
We also provide a high-throughput cloning service that follows the protocol outlined below.
- Preparation. We review the nucleotide sequence(s) that you provide to confirm the complete ORF sequence. Primers will be designed to flank the initiating methionine (ATG) and the stop codon. In cases where C-terminal fusions are desired, the open reading frames may be amplified without any stop codon (Figure 1).
- Amplification. Low-cycle, high-fidelity amplification of the desired ORF from a plasmid template.
- Screening. We will screen Thermo Fisher Scientific’s collection of full-length cDNA libraries if no appropriate plasmid template is available.
- Cloning. Our scientists will clone the amplified ORF into a Gateway Entry vector and transform the DNA into chemically competent E. coli (Figure 2).
- Sequencing. 5' and 3' end–read sequencing in order to verify amplification of the correct target as well as correct reading frame.
- Verification. Screening of antibiotic-resistant colonies to confirm positive Entry clones.
- Delivery. You receive the confirmed Entry clone(s) as glycerol stocks. You may have the Entry clone(s) transferred to one or more Gateway Destination vectors to create expression clones suitable for immediate application in the corresponding expression system(s).
Figure 2. High-Throughput ORF Cloning: Representative full-length gene expressed within an Invitrogen™ full-length cDNA library. Since the gene has been cloned into the pCMV•Sport 6.1 vector, it is flanked by the attB1 and attB2 Gateway recombination sites. The gene is composed of an open reading frame, illustrated as the area between the initiating methionine (ATG) and the stop codon. The open reading frame is that portion of the gene which codes for a functional protein. Flanking the open reading frame are 5´ and 3´ untranslated regions, which contain motifs involved in, for example, regulation, transcription, and posttranslational modifications.