GeneArt™ Type IIs Assembly Kit, Bbs I (Invitrogen™)

The GeneArt® Type IIs Assembly Kit, Bbs I, provides seamless cloning and assembly of up to 8 DNA fragments by simultaneous cleavage and ligation in a single reaction. The kit uses a technology similar to Golden Gate cloning with the type IIs restriction enzyme Bbs I and can be used to assemble multiple fragments in a pre-determined order into any compatible vector. Since type IIs assembly is not based upon homologous recombination, there is minimal risk of rearrangements and minimal sequence confirmation of your final construct is required.

The kit is offered in three versions, each with a different type IIs restriction enzyme (Aar I, Bsa I, or Bbs I). All versions include all-in-one enzyme mix, cloning vector, and cloning controls. Our GeneArt® Primer and Construct Design Tool should be used to determine the appropriate GeneArt® assembly kit for your fragments; it provides an easy-to-use interface to design your construct and create and order primers, should they be required.

• Assemble multiple DNA fragments in any order, into any compatible vector, without scars
• Avoid homologous recombination and associated rearrangements when cloning homologous or repetitive sequences
• Use for assembly of TALs, gene variants, and repetitive sequences
• Create your own cloning and expression vectors with custom vector elements
• Minimize sequence confirmation of final construct
• Pick from three type IIs enzymes

Type IIs Cloning
GeneArt® Type IIs Cloning is a simple, two-step process, consisting of an in vitro assembly reaction followed by transformation into competent E. coli. The type IIS restriction endonuclease Bbs I recognizes an asymmetric DNA sequence and cleaves the DNA molecule at a defined distance from the Bbs I recognition site. The ends of your DNA fragment can be designed to be flanked by the Bbs I restriction site such that digestion of the fragments removes the recognition site and generates complementary overhangs that can be ligated seamlessly, creating a junction that lacks the original site. The kit includes a destination vector, called pType IIs, which is optimized for this approach.

Cloning Efficiency, Flexibility, and Precision
With the GeneArt® Type IIs Assembly Kit, the main factors effecting cloning efficiency are the size of the DNA elements, the total size of the final molecule (? 10 kb), and the quality and specificity of the fragment.

Typical cloning efficiencies for different numbers of fragments cloned into pType IIs are:

• >95% for 5 fragments of 1 kb each
• >60% for 8 fragments of 1 kb each
• >85% for 2 identical fragments of 1 kb each

in silico Cloning Design Support
A key step in GeneArt® Type IIs Cloning is the correct design of fragments and oligos with the appropriate recognition sites and spacing to help ensure successful assembly of your clone. To simplify and speed the design process we provide the GeneArt® Primer and Construct Design Tool to help you design your experiment in silico. The tool recommends the correct GeneArt® kit for your assembly, checks for compatibility of the experimental design with the product specifications, designs DNA oligos, if needed, 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 VectorNTI® software.

Additional recommended products:

• Zero Blunt® TOPO® PCR Cloning Kit, without competent cells (Cat. No. 450245)
• Zero Blunt® TOPO® PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli (Cat. No. K280020)
• One Shot® MAX Efficiency™ DH10B™ T1 Phage Resistant Cells (Cat. No. 12331-013)