The GeneArt® High-Order Genetic Assembly System is a highly efficient kit for the simultaneous and seamless assembly of up to 10 DNA fragments, totaling up to 110 Kbp in length, into any vector. The system relies on yeast’s ability to take up and recombine DNA fragments with high efficiency. This greatly reduces the in vitro handling of DNA and eliminates the need for enzymatic treatments, such as restriction and ligation, while allowing for precise fusions of DNA sequences. The kit contains materials for the transformation and purification from yeast, including yeast selective media, and competent E. coli for plasmid amplification of correct clones.

• Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required
• Precision and Efficiency — Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind
• Flexibility — Use our linear vector, a vector of your choice, or clone pre-existing DNA fragments that have no end-homology without further modifications
• Free Tools — Design DNA oligos and more with our free web-based interface that walks you through your project step-by-step
• Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For the cloning of 1 to 4 DNA fragments of limited size, and if you prefer an in vitro approach, consider using the GeneArt® Seamless Cloning and Assembly Kit (cat # A13288).

Easily Create New Specific Constructs from Diverse DNA Fragments
The GeneArt® High-Order Genetic Assembly System takes advantage of transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisae to join pre-existing DNA fragments, or chemically synthesized oligonucleotides, into a single recombinant molecule. DNA fragments and linearized vector are joined based on shared end-terminal homology. If no such end homology exists between pieces, they can be “stitched" together with recombination linkers, synthetic DNA oligonucleotides that provide end-terminal homology between two unrelated DNA fragments. The process is very efficient and seamless, leaving no extra sequences after the assembly. Even though it has been shown to work for up to 0.5 Mb and 50 DNA fragments, this product has been optimized for up to 110 Kb and 10 DNA fragments in a vector.

Simple Clone Verification
In order to minimize the work that is done in yeast, recombinant yeast clones are subjected to a simplified 10-minute DNA extraction protocol. The extraction yields assembled molecule in enough quantity to do colony PCR verification of the junctions, as well as direct transformation of E.coli cells for downstream analysis. The proprietary lysis buffer and glass beads needed for the extraction are included in the kit.

Considerations for Choosing a Cloning Vector
The GeneArt® High-Order Genetic Assembly System requires shuttle vectors that have high capacity and are compatible with yeast and E.coli (i.e. BAC-YAC shuttle vectors). There is no cloning vector included in this product, but we offer a ready-to-use linear cloning vector separately called the GeneArt® pYES1L Vector with Sapphire Technology™ (cat# A13287). You can also use your own vector, but it must be compatible with the High-Order Genetic Assembly System. This compatibility can be easily accomplished with our GeneArt® High-Order Vector Conversion Cassette (cat#A13291).

Cloning Efficiency
In GeneArt® High-Order Assembly the main factors affecting cloning efficiency are the size of the DNA elements, the number of those without end-homology, the total size of the final molecule, and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency.

Typical cloning efficiencies for different numbers of fragments with end-homology assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ are the following:
• >90% for 5 DNA fragments of 10 Kb each
• >90% for 10 DNA fragments of 5 Kb each
• >50% for 10 DNA fragments of 10 Kb each

Common cloning efficiencies for pre-existing fragments, without end-homology, assembled into the GeneArt® pYES1L Vector with Sapphire Technology™ using 'stitching’ DNA oligonucleotides are:
• >90% for 1 fragment of 10 Kb
• >75% for 2 DNA fragments of 10 Kb each

Design Your Cloning in silico
A key step in GeneArt® High-Order Genetic Assembly is the correct design of fragments and oligos with the appropriate homology and spacing to ensure successful assembly of your clone. To simplify and speed the design process we provide a free online design tool to help you design your experiment in silico. The tool checks for compatibility of the experimental design with the product specifications, designs DNA oligos for either PCR amplification or for stitching of the different elements to clone, and presents the user with a graphical representation of the vector as well as a downloadable annotated sequence in GenBank format that is compatible with Vector NTI® software.

Applications
The GeneArt® High-Order Genetic Assembly System is designed to empower cloning and DNA assembly experiments in a wide range of molecular biology and synthetic biology applications, among others. The product allows for the creation of modular expression vectors, with interchangeable parts, and can be used to perform a variety of tasks that would otherwise involve multiple steps. Use the kit to simply: construct fusion proteins, delete, replace, or add DNA elements such as restriction sites, clone large pre-existing DNA fragments without end-homology, and many other techniques that require manipulation of genetic sequences.