GeneArt™ Seamless Cloning and Assembly Kit (Invitrogen™)

The GeneArt™ Seamless Cloning and Assembly Kit enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors.

• Speed and Ease — Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); 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
• Vector Flexibility — Use our linear vector or a vector of your choice
• Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project
• Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning more than 4 DNA fragments, final molecules larger than 110 Kb, or for using pre-existing DNA elements too long to be amplified by PCR; please consider the GeneArt™ High-Order Genetic Assembly System (cat# A13285).

Simple and Fast Clone Creation
GeneArt™ Seamless Cloning is a simple, two step process, consisting of a tube based assembly reaction followed by transformation into One Shot™ Chemically Competent TOP10 E. coli. The kit uses the properties of a proprietary enzymatic mix to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). A portion of the assembly reaction is then transformed into the provided competent TOP10 cells, yielding clones ready for analysis the next day. The required 15-base pair end-homology can be easily engineered by PCR-amplification with custom DNA oligos.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt™ Seamless Cloning and Assembly Kit the main factors effecting cloning efficiency are the size of the DNA elements (100 bp to 5 Kb), the total size of the final molecule (≤ 13 Kb), 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 cloned into pUC19L are the following:
• 90% for a single 5 Kb DNA element
• 70% for 4 fragments of 1 Kb each
• 40% for 4 DNA fragments of 2 Kb each

Success of the cloning is independent of the insert sequence and vector type, allowing you to design and add nearly any desired sequence, or combination of sequences, to any plasmid as long as it can be linearized by either restriction enzyme digestion or by PCR. The circularized clones obtain from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Cloning Design Support
A key step in GeneArt™ Seamless Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide a free online 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 with end-homology for the PCR amplification 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™ Seamless Cloning and Assembly Kit 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 in an existing vector, and many other techniques that require manipulation of genetic sequences.