GeneArt Type IIs cloning is a simple, two-step process based on the Golden Gate method, consisting of an in vitro assembly reaction followed by transformation into competent E. coli. Type IIs restriction endonucleases recognize asymmetric sequences and cleave these sequences at a defined distance from the recognition site. DNA ends can be designed to be flanked by a Type IIs restriction site such that digestion of the fragments removes the enzyme recognition sites and generates complementary overhangs. Such ends can be ligated seamlessly, creating a junction that lacks the original site or scars.
With GeneArt Type IIs kits you can:
- Assemble single or multiple (up to 8) DNA fragments in any order, into any compatible vector, without scars
- Avoid homologous recombination and associated rearrangements when cloning homologous or repetitive sequences
- Assemble GeneArt Strings DNA Fragments, GeneArt TALs Products and Services, gene variants, and repetitive sequences
- Create your own cloning and expression vectors with custom vector elements
- Minimize time and effort needed for sequence confirmation of final construct
- Perform your restriction/ligation reaction using any one of three Type IIs enzymes (AarI, BsaI, and BbsI); each 10-reaction kit contains all-in-one enzyme mix, cloning vector, and cloning controls
- Utilize the GeneArt Primer and Construct Design Tool for efficient assembly
Figure 1. Overview of the various cloning strategies possible when using GeneArt Type IIs Assembly Kits. (A) Diagram of the seamless assembly of two fragments using a combination of Type IIs restriction enzymes and ligase. The fragments and the corresponding sequences at their ends are shown in blue and green. Adaptors added to the fragments are shown in red. Underlined characters represent the recognition site for the Type IIs restriction endonuclease BsaI. (B) Diagram of two cloning scenarios: the cloning of two PCR fragments into a vector (left) and the transfer and assembly of fragments from two donor plasmids into a single vector (right). The black arrows indicate the orientation of the restriction sites, starting at the restriction site and pointing towards the cleavage sites. (C) Diagram illustrating the ease of transfer of a single fragment from a donor plasmid into multiple vectors with varying features (e.g., different vectors for bacterial vs. mammalian expression, RNAi expression, etc.). Arrows and abbreviations are as in (B). (D) Vector map of the recipient vector pType-IIs. Amp, ampicillin resistance gene; Kan, kanamycin resistance gene; CamR, chloramphenicol resistance gene; ccdB, ccdB counter-selectable marker.
Figure 2. GeneArt Type IIs Assembly kits with various types of DNA inserts. For each, cloning efficiency is defined as the correct insert(s) in the correct order and orientation, not the number of colonies. (A) Assembly of GeneArt Strings DNA Fragments, using AarI. (B) Assembly of PCR products, direct or precloned. (C) Assembly of repetitive (such as TALs or identical sequences) or small sequences.