The following vectors are used for inducible expression of HaloTag fusion proteins in E. coli and cell-free systems using the T7 RNA polymerase promoter. Expression levels depend highly on the nature of the protein, but in general the N-terminal HaloTag fusion protein (e.g., pFN18A/K, Cat.# G2751, G2681) can increase expression level, enhance refolding and boost solubility of the expressed protein. HaloTag vectors are supplied in two formats: as multiple cloning site (MCS) vectors for traditional cloning and as Flexi System vectors. The Flexi Vector System is a simple, directional cloning method for protein-coding sequences. It is based on two rare-cutting restriction enzymes, SgfI and PmeI, and provides a rapid, efficient and high-fidelity way to transfer protein-coding regions between a variety of Flexi Vectors without the need to resequence. Direct transfers can only occur between two N-terminal tagged vectors or from an N-terminal to a C-terminal vector. The MCS vectors and several Flexi system vectors contain a His6-HaloTag dual tag. The dual tag enables protein purification with the reusable Ni-resin while retaining the HaloTag covalent labeling properties. Multiple Cloning Site (MCS) Vectors: pH6HTN His6HaloTag T7 Vector (Cat.# G7971) is designed for protein expression with an N-terminal His6-HaloTag dual tag in E. coli and T7 cell-free expression systems. pH6HTC His6HaloTag T7 Vector (Cat.# G8031) is designed for protein expression with a C-terminal His6-HaloTag dual tag in E. coli and T7 cell-free expression systems. Flexi System Vectors: pF1A/K T7 Flexi Vectors (Cat.# C8441, C8451) are designed for untagged protein expression. pFN18A/K HaloTag T7 Flexi Vectors (Cat.# G2751, G2681) are designed for protein expression with an N-terminal HaloTag in E. coli and T7 cell-free expression systems. pFN19A/K HaloTag T7 SP6 Flexi Vectors (Cat.# G1891, G1841) are designed for protein expression with an N-terminal HaloTag in T7 and SP6 cell-free expression systems. These vectors are optimized for cell-free expression systems. pFC20A/K HaloTag T7 SP6 Flexi Vectors (Cat.# G1681, G1691) are designed for protein expression with a C-terminal HaloTag in E. coli and SP6 cell-free expression systems. These vectors are optimized for cell-free expression systems. pFN29A/K His6HaloTag T7 Flexi Vectors (Cat.# G8261, G8331) are designed for protein expression with an N-terminal His6-HaloTag dual tag in E. coli T7 cell-free expression systems. pFC30A/K His6HaloTag T7 Flexi Vectors (Cat.# G8321, G8381) are designed for protein expression with a C-terminal His6-HaloTag dual tag in E. coli T7 cell-free expression systems. Note: Flexi Vectors contain the lethal barnase gene to reduce background colonies without inserts during the subcloning procedure. Using the Flexi Vector Cloning System replaces the barnase gene with your insert. These vectors, as purchased, cannot be cultured in normal laboratory strains of E. coli without an insert.|
Choice of Systems: Choose between traditional (MCS) and Flexi(R), cloning to get the benefits of HaloTag(R), technology. Dual Tag: Couple the protein solubility and labeling benefits of HaloTag(R), technology with the reusability and the throughput of Ni-affinity technology. Versatile Cloning: Choose from a variety of expression systems and fusion tag orientations and then transfer to others as required (for Flexi(R), system). Time Savings: Barnase insert (Flexi(R), system) decreases the number of background colonies, allowing efficient transfer of genetic constructs.