Like our first generation Seamless Cloning and Assembly Kit, GeneArt™ Seamless PLUS Cloning and Assembly Kit is the complete kit for simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute or less, room temperature reaction. However, Seamless PLUS offers several advantages over previous kits:

• Increased Efficiency: pre-cloning option for large fragments for increased cloning efficiency
• Larger Constructs: create constructs up to 40 kb
• Versatility: high capacity, broad-range conjugative vector that replicates in most Gram negative bacteria

The improvements above are combined with these key benefits shared by all GeneArt™ Seamless Cloning and Assembly kits:

• Speed and Ease—clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector; 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
• Free Tools—design your final construct and DNA oligos in silico using our free web-based tool that takes you step-by-step through your project
• Vector Flexibility—use our linear vector or a vector of your choice
• Diverse Applications—streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments

For cloning of more than 4 DNA fragments or for final molecules larger than 110 Kb, please consider the GeneArt™ High-Order Genetic Assembly System.

Simple and Fast Clone Creation
GeneArt™ Seamless PLUS Cloning is a simple, two-step process, consisting of in vitro assembly followed by transformation into One Shot™ DH10B™ T1R SA competent E. coli. The kit employs a proprietary enzyme/buffer mix to assemble DNA fragments with shared terminal end homology without extra sequences or scars in the final construct (“seamless”). Terminal end homology is easily incorporated by PCR amplification with custom DNA oligos engineered using our free web tool.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt™ Seamless PLUS Cloning and Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA fragments (100 bp to 10 Kb), the total size of the final molecule (≤ 40 Kb), and the quality and specificity of each fragment.

Typical cloning efficiencies for different numbers of fragments:

• >95% for 4 fragments, 5 Kb each
• >90% for 4 fragments, 10 Kb each

Cloning success 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 PCR. The circularized clones obtained from the reaction contain only the sequence of your original vector, inserts, and designated homologies, with no extraneous nucleotide insertions.

in silico Design Support
A key step in GeneArt™ Seamless PLUS Cloning is the correct design of fragments and oligos with the appropriate homology and spacing to help ensure successful assembly of your clone. We provide a free online tool, the GeneArt™ Design Tool for Seamless or High-Order Assembly and Mutagenesis, 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 PLUS Cloning and Assembly Kit is designed to empower cloning and DNA assembly 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 construct fusion proteins; delete, replace, or add DNA elements such as restriction sites in an existing vector; and carry out many other techniques that require manipulation of genetic sequences.