The Gateway Cloning System Introduction to the Gateway

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The Gateway® Cloning System Introduction to the Gateway® System Contents • Defining Gateway® technology.

The Gateway® Cloning System Introduction to the Gateway® System Contents • Defining Gateway® technology. • Advantages of Gateway® cloning. • Ways to enter the Gateway® system. • How to obtain a Gateway® expression clone.

The Gateway® Cloning System PCR Your Source Library ORF collection Gene synthesis Gene Protein

The Gateway® Cloning System PCR Your Source Library ORF collection Gene synthesis Gene Protein Localization Your Application Gene Entry Clone Gene Protein Purification Directional cloning • Maintains reading frame • No restriction enzymes • No ligation • 1 hour, roomtemperature reaction with >99% efficiency • No re-sequencing • Compatible with automation • Reversible reactions RNAi Gene Cell-Free Protein interaction Gene 1 Gene 2 Gene 3 Your Application 2 • Gene 4

Key Benefits of the Gateway® Technology · Efficiently and easily shuttle insert DNA from

Key Benefits of the Gateway® Technology · Efficiently and easily shuttle insert DNA from one expression plasmid to another · Simplify the cloning workflow and save time · Create expression clones without using restriction enzymes and ligase · Utilize Ultimate™ ORF clones, a pre-made Gateway® collection · Simultaneously clone, in a specific order and orientation, up to 4 DNA fragments into one plasmid 3

Phage lambda recombination in E. coli cos Phage l The Gateway® System relies att.

Phage lambda recombination in E. coli cos Phage l The Gateway® System relies att. P on five sets of specific and 232 bp non cross-reacting att x sequences att. B E. coli 21 bp Integration (Int, IHF) Excision (Int, IHF, Xis) The specificity is given by the 7 nucleotides of the core region Lysogen 4 att. L att. R 96 bp 157 bp

Building a Gateway® Entry Clone + + BP Clonase™ II 90 -99% correct clones

Building a Gateway® Entry Clone + + BP Clonase™ II 90 -99% correct clones on Kan plates 5

Obtaining a Gateway® Expression Clone + + LR Clonase™ II 90 -99% correct clones

Obtaining a Gateway® Expression Clone + + LR Clonase™ II 90 -99% correct clones on Amp plates 6

Number of publications each year Gateway® Cloning Platform – a Proven Technology 800 700

Number of publications each year Gateway® Cloning Platform – a Proven Technology 800 700 600 500 400 300 200 100 0 1 2 3 4 5 6 Years after Gateway® Technology was introduced John M. Watson, et al. FEBS Letters – “The Gateway® System seems to have become a universal standard in genomic research and there are many gene and gene fragment libraries in these Entry vectors. “ 7

Glossary of terms used in Gateway® cloning att site – A defined length of

Glossary of terms used in Gateway® cloning att site – A defined length of DNA that constitutes a recombination site. There are 4 classes of att sites called att. B, att. P, att. L, and att. R. ccd. B gene – A counterselectable gene that allows for negative selection of unwanted byproduct plasmids after recombination. Donor (p. DONR) Vector – A vector with att. P sites flanking a counterselectable gene that recombines with a gene of interest flanked by att. B sites. BP reaction – A recombination event between att. B and att. P sites catalyzed by BP Clonase™ II Entry (p. ENTR) clone – A vector that contains your gene of interest flanked by att. L or att. R sites. LR reaction – A recombination event between att. L and att. R sites catalyzed by LR Clonase™ II Destination (DEST) Vector – An application-geared vector with att. R sites flanking a counterselectable gene that will recombine with one or more entry clones. Multi. Site Gateway® Technology – A system that allows simultaneous assembly of multiple DNA fragments into a single destination vector 8

Policy for the use and distribution of Gateway® Technology The Gateway® Technology is covered

Policy for the use and distribution of Gateway® Technology The Gateway® Technology is covered under Limited Use Label License. For more information regarding use, please see LULL no. 19 at www. invitrogen. com. Invitrogen offers distribution of Gateway® entry clones as follows: • Gateway® entry clones, containing att. L 1 and att. L 2 sites, may be generated by academic and government researchers for the purpose of scientific research. Such clones may be distributed for scientific research by non-profit organizations and by for-profit organizations without royalty payment to Invitrogen. • Gateway® expression clones, containing att. B 1 and att. B 2 sites, may be generated by academic and government researchers for the purpose of scientific research. Such clones may be distributed for scientific research by academic and government organizations without royalty payment to Invitrogen. Organizations other than academic and government may also distribute such Gateway® expression clones for a nominal fee payable to Invitrogen. • For inquiries, please contact Invitrogen’s licensing department. 9