DNA Component Explanation

Our plasmids are composed of both a core vector backbone and a series of DNA components. Our plasmid platform is all built around the same core backbone, which means that the DNA components within each plasmids can be interchanged into each other. Below follows a brief introduction to what we consider a DNA component in our plasmids to be.

For the purposes of our website we define a DNA component as a region of DNA that has a useful research purpose and is flanked by two restriction sites that make compatible with the core plasmid structure on which all of our vectors are based. So for example, the coding sequence for the reporter gene luciferase in itself would not described on our site as a component on its own, however, if we have inserted into one of our plasmids and it is now flanked by two restriction sites that allow it to be expressed in a particular configuration, then we would describe it as a DNA component. Such components can then be purchased though this website as they are required.

This definition does not mean that a component only contains one DNA sequence of interest. An entire expression cassette can also be considered as a component on this site, provided that the entire cassette is flanked by two sites that are compatible with our plasmids, and that the entire DNA stretch does not contain other restriction sites that conflict with our system within it. So for example, we have many reporter gene expression cassettes that contain a promoter, gene, and terminator sequence, where the entire region is flanked by two sites and contains no other sites within it (see figure 1).

Details about individual components

• Mammalian Antibiotic Selection Options
• Bacterial Antibiotic Selection Options
• Secretory tags/signal peptides
• Bacterial Secretory Tag (Signal Peptide) Plasmids
• Yeast Secretory Tag (Signal Peptide) Plasmids
• Mammalian Secretory Tag (Signal Peptide) Plasmids
• Protein & Peptide Tags
• Origin of Replication Options
• Multiple Cloning Sites / Polylinkers Options
• Promoters
• Transcriptional Terminators

Mammalian Antibiotic Selection Options

We currently sell five alternative mammalian antibiotic selection gene options. These have been inserted into four configurations within our plasmids to allow you to choose the selection system you would prefer. These include:

• Under the direct control of the CMV promoter in the main MCS.
• Under control of the PGK promoter immediately after the main multiple cloning site. The poly-adenylation signal is the same as that used by the upstream CMV promoter.
• Under the control of an internal ribosome entry site (EMCV or FMDV). These can often demonstrate low, or variable activity depending on the sequence, cell line and sequence of the upstream gene. Yields of 1-5% of the upstream are not uncommon.
• Under the control of either the Rous Sarcoma Virus (RSV) promoter (low expression) or the Human Ubiquitin (Ub) promoter (high expression), where the entire expression cassette is inserted away from the main multiple cloning site in the other half of our vector, flanked by two AscI restriction sites.

Mammalian Antibiotic Selection Gene Options

We currently sell five different reporter genes, including:

• Puromycin
• Hygromycin
• Blasticidin
• Neomycin / G418
• Zeocin

Puromycin-Resistance Plasmids

Puromycin is an aminonucleoside antibiotic. It is produced naturally by the Streptomyces alboniger bacterium and disrupts peptide transfer on ribosomes causing premature chain termination of protein synthesis. It is a powerful inhibitor of translation in both prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the puromycin N-acetyl-transferase gene (pac), also from Streptomyces.

The pac gene is readily include into plasmids and provides a simple selectable marker, most commonly used in mammalian cells and yeast. For isolation of puromycin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum puromycin concentration, typically 1-10 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

1. Following transfection (24-48h) incubate cells in fresh medium containing Puromycin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.
2. Remove and replace the puromycin-containing medium every 3-4 days.
3. Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.
4. Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Hygromycin-Resistance Plasmids

Hygromycin B is an aminoglycoside antibiotic isolated from Streptomyces hygroscopicus. It disrupts translation byinterfering with translocation and causes mistranslation at the 70S ribosome. Hygromycin B kills most bacteria, fungi and higher eukaryotes.  Resistance to hygromycin is conferred by the hph gene from E. coli. Hygromycin B is normally used at concentrations of 50-200 µg/ml in eukaryotic cells.

The 1 kb hph gene is readily include into plasmids and provides a simple selectable marker, most commonly used in mammalian cells and yeast. For isolation of hygromycin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum hygromycin concentration, typically 50-200 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

1. Following transfection (24-48h) incubate cells in fresh medium containing hygromycin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.
2. Remove and replace the hygromycin-containing medium every 3-4 days.
3. Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.
4. Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Blasticidin-Resistance Plasmids

Blasticidin is a nucleoside antibiotic produced by the bacterium Streptomyces griseochromogenes. It is a powerful inhibitor of translation in both prokaryotic and eukaryotic cells. Resistance is conferred by the bsd gene from Aspergillus terreus. Blasticidin acts quickly and causes cell death at low concentrations. E. coli strains are generally killed by concentrations of 50 µg/ml, while mammalian cells are killed by concentrations as low as 2 to 10 µg/ml.

The 393 bp bsd gene encodes a polypeptide of 130 amino acids. It is readily included into plasmids and provides a simple selectable marker, most commonly used in mammalian cells and yeast. For isolation of blasticidin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum blasticidin concentration, typically 2-20 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

1. Following transfection (24-48h) incubate cells in fresh medium containing blasticidin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.
2. Remove and replace the blasticidin-containing medium every 3-4 days.
3. Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.
4. Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Zeocin-Resistance Plasmids

Zeocin is a copper chelated glycopeptide antibiotic related to bleomycin and causes cell death by intercalating into DNA and cleaving it. It is very toxic to a range of organisms, including mammalian and insect cells, as well as yeast, bacteria, and plants.  Resistance to Zeocin™ is conferred by the Sh ble gene, which binds and prevents it from interacting with DNA. The broad applicability of Zeocin resistance means that a single selectable marker can be used for several different cell types. E. coli strains are generally killed by concentrations of 25 µg/ml, while mammalian cells are killed by concentrations 50 - 400 µg/ml.

The 370 bp Sh ble gene is readily included into plasmids and provides a simple selectable marker, for many different cell types. For isolation of Zeocin-resistant mammalian cells, preliminary experiments should be conducted to select the optimum Zeocin concentration, typically 25 - 400 µg/ml, depending on the cells used.

A typical selection protocol includes the following key steps:

1. Following transfection (24-48h) incubate cells in fresh medium containing Zeocin at the appropriate concentration. The selection process will work best if cells are actively dividing, and not confluent.
2. Remove and replace the Zeocin-containing medium every 3-4 days.
3. Examine cells for the formation of foci after 7 days of selection. Formation of foci may require several additional days to develop depending on the host cell line and transfection/selection efficiency.
4. Transfer and pool resistant clones to a fresh cell culture plate and maintain on selection medium for another week. This pooled culture provides the source of resistant cells.

Bacterial Antibiotic Selection Options

Our bacterial selection markers are normally flanked by two PmeI restriction sites. They will function if they are inserted in either orientation between these two sites. We currently stock four selectable markers flanked by PmeI sites to be used in bacteria including:

• Ampicillin / Carbenicillin
• Kanamycin / Neomycin
• Spectinomycin / Streptomycin
• Chloramphenicol

Reporter Genes:

Cloning options: We sell our reporter genes in a range of configurations, including:

• Under the direct control of a promoter upstream of the reporter gene in the main MCS.
• Under control of the PGK promoter for expression in mammalian cells immediately after the main multiple cloning site. The poly adenylation signal is the same as that used by the upstream promoter (normally CMV in these plasmids).
• Under the control of an internal ribosome entry site (EMCV or FMDV) for expression in mammalian cells. These can often demonstrate low, or variable activity depending on the sequence, cell line and upstream gene. Total protein content yields of 3-5% of the upstream gene are not uncommon, although 40-60% of cells may register as positive by transfection, depending on the reporter gene and the assay.
• Under the control of either the Rous Sarcoma Virus (RSV) promoter (low expression) or the Human Ubiquitin (Ub) promoter (high expression), where the entire expression cassette is inserted away from the main multiple cloning site, flanked by two AscI restriction sites.

Reporter Gene Options:

We have six different reporter genes, including:

• Photinus pyralis luciferase (FLuc, pGL4 derived) - the brightest and most up to date version of this gene.
• Renilla reniformis luciferase (RLuc). Slightly smaller than FLuc and uses a different substrate.
• daGFP - a small, bright, synthetic fluroescent gene with similar properties to GFP.
• Beta Galactosidase (β-Gal)
• Human Secreted Alkaline Phosphatase (SEAP)
• Chloramphenicol Acetyl Transferase (CAT)

Firefly Luciferase (Photinus) Plasmid Vector Information

The name ‘luciferase’ is derived from Lucifer or 'light-bearer'. Firely luciferase is a commonly used reporter gene with high specific luminescence. It is an enzyme that catalyses the production of light by combining luciferin with ATP.

luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP + light

The activity of luciferase is readily quantified in luciferase assays, and the luminescence colour can vary between yellow-green (λmax = 550 nm) to red (λmax = 620). The molecule contains 550 amino acids, with a molecular weight of 62 KDa. A typical luciferase assay includes the following key steps:

• Introduction of luciferase expression plasmid into cells by transfection or infection
• Incubation for desired time period, then lysis of cells (typically after 24-48h)
• Addition of luciferin substrate and ATP
• Measure light emission using a luminometer

Firefly luciferase sequence

MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSL
QFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPP
GFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRV
VLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILI
TPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFI
VDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFV
DEVPKGLTGKLDARKIREILIKAKKGGKIAV-

Renilla (Sea Pansy) Luciferase Plasmids

The name ‘luciferase’ is derived from Lucifer or 'light-bearer'. Renilla luciferase is a commonly used reporter gene with high specific luminescence, derived from the soft coral coelenterate Renilla reniformis. It is an enzyme that catalyses the production of light by oxidising coelenterazine.

coelenterazine + O₂ → coelenteramide + CO₂ + photon of light

The activity of luciferase is readily quantified in luciferase assays. The molecule contains 331 amino acids. A typical luciferase assay includes the following key steps:

• Introduction of luciferase expression plasmid into cells by transfection or infection
• Incubation for desired time period, then lysis of cells (typically after 24-48h)
• Addition of coelenterazine substrate and oxygen
• Measure light emission using a luminometer

Beta-galactosidase Plasmid Vectors Information

Beta-galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. It is a commonly used and versatile reporter gene, suitable for producing an enzymatic colour change during histology and also for activating fluorescent prodrugs for visualisation in flow cytometry.

Beta-galactosidase also provides the basis for the blue/white screening of recombinant clones. This approach relies on the enzyme being divided into two inactive peptides, LacZα and LacZΩ. Enzyme activity is restored only when both are present in the same cell, as they spontaneously reassemble into a functional enzyme. This property is exploited in many cloning systems where the presence of the lacZα gene can complement another mutant gene encoding the LacZΩ. The presence of active β-galactosidase is detected using X-gal, which produces a characteristic blue colour when cleaved by β-galactosidase, thereby providing an easy means of distinguishing the presence of cloned product in a plasmid. β-galactosidase from E. coli has 1024 amino acids and forms a 464 kDa homotetramer.

A typical beta galactosidase assay includes the following key steps:

• Introduction of beta galactosidase expression plasmid into cells by transfection or infection
• Incubation for desired time period, then lysis of cells (typically after 24-48h)
• Addition of substrate, typically X-gal for colour change reaction
• Stop the reaction when adequate colour change has occurred, and read using spectrophotometer at 420 nm.

SEAP (SEcreted Alkaline Phosphatase)

Alkaline phosphatase is a metalloenzyme capable of removing phosphate groups from a variety of substrates under alkaline conditions, in the presence of magnesium and zinc ions. It provides a simple and versatile reporter gene, used in histology as well as to measure expression from plasmid vectors.

Oxford Genetics encodes human alkaline phosphatase as a reporter gene, including a C-terminal 24 amino acid-truncated version which is secreted from cells and provides a useful non-destructive reporter gene for use both in vitro and in vivo.

Alkaline phosphatase is typically measured in colour change reactions, for example the hydrolysis of para-nitrophenylphosphate disodium salt (PNPP), which yields a distinct colour change that can be measured at 405 nm. Our secreted human alkaline phosphatase contains 1566 base pairs.

A typical alkaline phosphatase assay includes the following key steps:

• Introduction of alkaline phosphatase expression plasmid into cells by transfection or infection
• Incubation for desired time period, then lysis of cells (typically after 24-48h) or harvesting of supernatant (for SEAP)
• Addition of substrate, typically PNPP for colour change reaction
• Stop the reaction when adequate colour change has occurred, and read using spectrophotometer at 405 nm.

Secretory tags/signal peptides

Our Secretion Plasmids

We sell a range of secretory signal peptide plasmids that allow the export of a protein from the cytosol into the secretory pathway. Proteins can exhibit differential levels of successful secrection and often certain signal peptides can cause lower or higher levels when partnered with specific proteins. For this reason we sell 10 signal peptides for secretion in mammalian cells, 10 for secretion in bacterial cells and 6 for secretion from yeast cells. This provides a range of plasmid options to enable the successful secretion of your proteins.

In eukaryotes the signal peptide is a hydrophobic string of amino acids that is recognised by the Signal recognition particle (SRP) in the cytosol of eukaryotic cells. After the signal peptide is produced from a mRNA-ribosome complex, the SRP binds the peptide and stops protein translation. The SRP then shuttles the mRNA/ribosome complex to the rough endoplasmic reticulum where the protein is translated into the lumen of the endoplasmic reticulum. The signal peptide is then cleaved off the protein to produce either a soluble, or membrane tagged (if a transmembrane region is also present), protein in the endoplasmic reticulum.  Signal peptides contain the sequences that are responsible for their own cleavage. This cleavage point will be highlighted in each individual product data sheet.

In prokaryotes, the most commonly used secretory tags are the OmpA and PelB secretion tags. These signals peptide function similarly to their eukaryotic counterpart, however, because prokaryotes have no internal membranous organelles, and bacteria have either a cell wall (gram-positive) or a second membrane and a cell wall (gram-negative), the protein is normally secreted into the periplasmic space rather than the supernatent.

Bacterial Secretory Tag (Signal Peptide) Plasmids

Oxford Genetics provides versatile cloning plasmids containing the following bacterial secretory tags. Simply inserting your gene within the MCS will introduce the secretory tag onto the N terminus, and allow it to be automatically removed by cleavage during secretion:

• OmpA (outer membrane protein A)
• OmpC (outer membrane protein C)
• OmpT (outer membrane protein T)
• PelB (pectate lyase B)
• TorA (tor operon component)
• TorT (tor operon component)
• MalE
• Dsba (dithiol-disulfide oxidoreductase)
• gIII (M13 phage minor coat protein)
• PhoA (bacterial alkaline phosphatase)
• Sufl

Simple constructs

Each of these proteins is provided in a vector allowing N-terminal fusion with your gene of interest by inserting it into the MCS. The tag will be automatically removed by cleavage during secretion. The vectors usually also include kanamycin resistance to enable simple selection of transfected bacterial cells.

Constructs containing Secretory Peptides and Additional Functional Tags

We have had particular success using the OmpA and PelB secretory tags, and we provide the world’s most extensive range of products using those particular tags in a very broad range of formats.

For example both OmpA and PelB-containing plasmids are provided with additional epitope and affinity tags, allowing simple identification and/or purification of your expressed proteins. These tags include hexahistidine and decahistidine, V5, C-Myc, T7, haemagglutinin and several others.

Constructs containing Secretory Peptides, Additional Functional Tags and Enzyme Cleavage Sites Enabling Removal of Functional Tags After Protein Production

Many of our plasmids that contain functional tags (eg. Epitope tags, in addition to secretory tags) also contain simple cleavage sites. This allows you to remove the tag, if you so desire, following protein production and identification/purification. We sell a broad range of cleavage sites, including sites for EKT, Factor Xa, Thrombin, TEV, 3C (Prescission) etc Because we provide a large number of products with three components (secretory tag, additional functional tag and cleavage site) we have tabulated them below to help you identify which plasmid you need.

For simplicity, all of our tags are positioned upstream of the MCS and will be included at the N terminal of your inserted protein sequence. This should be sufficient for all needs, however do contact us if you require C terminal tags, as these are very easy for us to provide.

Yeast Secretory Tag (Signal Peptide) Plasmids

We offer several different secretory tags for use in yeast cells, in easy-to-clone formats. The rationale for selling several different tags is because experience shows that no single tag is ideal for all proteins, and some proteins work better with different secretory tags. Oxford Genetics provides the following secretory tags for use in yeast cells:

• Inulase
• Invertase
• Killer protein
• Lysozyme
• Albumin
• Alpha amylase
• Alpha factor (full length)
• Alpha factor (secretory peptide)
• Glucoamylase

Each of these secretory peptides is provided in a vector allowing simple N-terminal fusion with your gene of interest. Most of the constructs also include the auxotrophic Uracil selection expression cassette URA3 gene, allowing positive selection of transfected yeast cells by growing the population in the absence of uracil. We also provide the alpha amylase secretory peptide and the full length alpha factor in a comprehensive range of formats to meet all you requirements:

Alpha amylase signal peptide and the full length alpha factor; we sell an extensive range of plasmids containing these signal peptides together with a range of peptide tags and enzyme-cleavage sites. In each case the secretory signal peptide will be naturally removed during secretion and the enzyme cleavage site allows removal of the tag from the protein if/when required after translation.

We provide these secretory signal peptides with many different epitope or, affinity tags

• Epitope tags: V5, S-Tag, C-Myc, FLAG, Haemagglutinin (HA), T7
• Affinity tags: streptavidin, hexahistidine

Combined with a broad range of enzyme cleavage sites:

• Factor Xa
• Enterokinase (EKT)
• 3C Prescission
• Thrombin
• TEV (Tobacco Etch Virus)

Mammalian Secretory Tag (Signal Peptide) Plasmids

We offer several different mammalian secretory tags in easy-to-clone formats. The rationale for selling several different tags is because experience shows that no single tag is ideal for all proteins, and some proteins work better with different secretory tags.

Oxford Genetics provides the following mammalian secretory tags:

• BM-40 (osteonectin SPARC)
• Vesicular Stomatitis Virus G (VSVG) protein
• Chymotrypsinogen
• Human interleukin-2 (IL-2)
• Gaussia luciferase
• Human serum albumin
• Influenza haemagglutinin
• Human insulin

Each of these proteins is provided in a vector allowing simple N-terminal fusion with your gene of interest. Most of the constructs also include puromycin resistance to enable simple selection of transfected cells. We also provide the insulin secretory peptide and the BM-40 secretory peptides in more sophisticated formats, providing the world's largest range of combinations of signal peptides with additional functional tags and enzyme cleavage sites:

BM-40 (osteonectin SPARC) and Insulin secretory signal peptides; we sell an extensive range of plasmids containing these signal peptides together with various peptide tags and enzyme-cleavage sites. In each case the secretory signal peptide will be naturally removed during secretion and the enzyme cleavage site allows removal of the tag from the protein if/when required after translation.

We provide these secretory signal peptides with many different epitope or affinity tags

• Epitope tags: V5, S-Tag, C-Myc, FLAG, Haemagglutinin (HA), T7
• Affinity tags: Streptavidin, hexahistidine

Combined with a broad range of enzyme cleavage sites:

• Factor Xa
• enterokinase (EKT)
• 3C Prescission
• Thrombin
• TEV (tobacco Etch Virus)

Protein & Peptide Tags

We currently have plasmids that contain the following peptide tags:

• Histidine (6 His)
• Histidin (10 His)
• Influenza HA
• C-Myc
• FLAG
• Strep
• V5
• Glutathione-S-transferase (GST)
• Maltose binding protein (MBP)
• Vesicular Stomatitis Vrisu G Epitope
• T7
• HSV
• E-Tag
• S-Tag
• Thioredoxin (TRX)

We also provide a range of reporter gene tags including Renilla and Firefly luciferase, Beta Galactosidase, Alkaline Phosphatase, Chloramphenicol Acetly transferase and a range of fluorescent reporters. In addition, all of our peptide tags are available with five different protease cleavage tags including:

• Enterokinase (EKT)
• Rhinovirus 3C (PreScission)
• Tobacco Etch Virus (TEV)
• Thrombin
• Factor Xa

c-Myc tag

c-Myc tags are decapeptide tags derived from the c-myc gene product that can be encoded within DNA sequences and be positioned at either end of the protein of interest. C-Myc tags can be used for affinity purification, co-precipitation and also as epitope tags for detection by western blotting. Anti-c-Myc antibodies are efficient for detection in a range of formats. The c-Myc tag sequence, from N to C: N-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-C. It has a molecular weight of 1202 Da.

A typical c-Myc immunoprecipitation is as follows:

1. Harvest and lyse host cells with expressed c-Myc-tagged protein, and clarify by centrifugation
2. Incubate the lysate with anti-a-Myc antibodies and a slurry of Protein A-capture gel beads, centrifuge and wash.
3. Elute the bound c-Myc-tagged protein with one of (a) a low pH glycine buffer, (b) SDS, (c) high pH urea buffer.

FLAG Tag Plasmids

FLAG^® tags are octapeptide tags that can be encoded within DNA sequences and be positioned at either end of the protein of interest. FLAG tags can be used for affinity purification, co-precipitation and also for detection by western blotting. The FLAG tag is synthetic, and was designed to be small and hydrophilic, to exert minimal effects on the structure of proteins it is linked to. Anti-FLAG antibodies are efficient for detection in a range of formats. The FLAG tag also contains an intrinsic enterokinase (EKT) cleavage site, for tag removal after it has fulfilled its purpose. The FLAG tag sequence, from N to C: DYKDDDDK . It has a molecular weight of 1012 Da.

A typical FLAG immunoprecipitation is as follows:

1. Harvest and lyse host cells with expressed FLAG-tagged protein, and clarify by centrifugation
2. Incubate the lysate with a slurry of FLAG-capture gel beads, centrifuge and wash carefully.
3. Elute the bound FLAG-tagged protein and its binding partner(s) with a buffer containing the free FLAG-elution peptide DYKDDDDK.

GST Tag Plasmids

Glutathione-S-transferase tags (GST tags) can be encoded within DNA sequences and be positioned at either end of the protein of interest, creating a fusion protein. GST tags are widely used affinity and solubility tags, they can be used for solubilisation, purification and also for detection by western blotting. Purification normally involves adsorption of the protein to GSH-containing beads, followed by washing to remove impurities and then displacement of the protein of interest using free GSH. In addition anti-GST antibodies are efficient for detection in a range of formats. Because GST activity is enzymatic, it works best for systems where the protein of interest is purified without denaturation.

GST contains 220 amino acids and has a molecular weight of 26 KDa.

A typical GST purification is as follows:

1. Harvest and lyse host cells with expressed GST-tagged protein, and clarify by centrifugation
2. Incubate the lysate with a slurry of GSH-bearing beads, wash and pack the slurry into an empty disposable PD10 column.
3. Wash and elute the bound GST-tagged protein with a buffer containing excess GSH.

His Tag Plasmids

Histidine tags (His tags) can be easily encoded within DNA sequences and be positioned at either end of the protein. They can be used both for purification and also for detection by western blotting. Traditionally purification has involved adsorption of the protein to nickel or cobalt chromatography columns, followed by elution with a base. However anti-His antibodies are efficient for detection and can also be used successfully for purification in a range of formats.

Most his tags are hexahistidine (His6) although His10 is also available, and binds more strongly to the nickel and cobalt columns. His tags are suitable for purification of many proteins, including those that need to be purified denatured. Histidine has a weak positive charge at neutral pH. His6 has a molecular weight of 930 Da, while His10 is 1550 Da

A typical hexahistidine purification is as follows:

1. Harvest and lyse host cells with expressed histidine-tagged protein, and clarify by centrifugation.
2. Incubate the lysate with a slurry of Nickel beads (eg Ni-Sepharose). I ml of 50% slurry can bind about 20 mg of His-tagged protein
3. Pack the slurry into an empty disposable PD10 column

Maltose-Binding Protein (MBP) Tag Plasmids

The MBP tag is derived from the E. coli maltose/maltodextrin system and is widely used as both a solubility tag (notably for recombinant proteins grown in E. coli) and also as an affinity tag, since it binds to amylose columns. MBP has a molecular mass of 42.5 KDa.

A typical MBP purification is as follows:

• Harvest and lyse host cells with expressed MBP-tagged protein, and clarify by centrifugation
• Pass the lysate slowly through an amylose column and wash carefully with buffer.
• Elute the bound MBP-tagged protein and its binding partner(s) with a buffer containing high levels of free maltose
• Purify by dialysis.

S-Tag Plasmids

The S-tag is derived from the N terminus of RNase A. It is a 15 amino acid sequence that is used as an epitope tag and also as a solubility tag. The sequence is: Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser. S-tag is suitable for use in many different cell types and has a molecular mass of 42.5 KDa.

A typical S-tag-based purification is as follows:

1. Harvest and lyse host cells with expressed S-tagged protein, and clarify by centrifugation
2. Pass the lysate slowly through an affinity column containing anti-S-Tag antibodies and wash carefully with buffer.
3. Elute the bound S-tagged protein and its binding partner(s) with a buffer containing high levels of free S-Tag peptide

Thioredoxin (TRX) Tag Plasmids

The Thioredoxin (TRX) tag is used predominantly to increase the solubility and thermal stability of proteins expressed in bacterial systems, where it also assists in the refolding of proteins requiring a reducing environment. It also provides a useful epitope tag, and has a molecular weight of approximately 12 KDa.

A typical TRX protein production is as follows:

1. Harvest and lyse host cells with expressed TRX-tagged protein, and clarify by centrifugation
2. TRX and many TRX fusion proteins are stable at 80˚C, allowing initial purification by heat treatment to precipitate other proteins.
3. Several types of affinity purification are possible, for example where an immobilized arsenical compound forms an adduct with the redox-sensitive vicinal dithiols present at the active site of thioredoxin. Pass the lysate slowly through an affinity column and wash carefully with buffer
4. Elute by changing the pH and reducing potential of the buffer
5. Purify by dialysis.

V5 Tag Plasmids:

The V5 epitope tag is derived from a peptide present at the C terminus of the P and V proteins of simian virus 5. It contains 14 amino acids, from N to C: GKPIPNPLLGLDST.

A typical V5 immunoprecipitation is as follows:

1. Harvest and lyse host cells with expressed V5-tagged protein, and clarify by centrifugation
2. Incubate the lysate with a slurry of gel beads bearing V5-capture antibodies, centrifuge and wash carefully.
3. Elute the bound V5-tagged protein and its binding partner(s) with a buffer containing the free V5-elution peptide GKPIPNPLLGLDST.

Origin of Replication Options

Bacterial Origin Cloning: We currently have four alternative origins of replication for use in bacteria. Our bacterial origins of replication are normally flanked by two SwaI restriction sites. They will function if they are inserted in either orientation between these two sites.

Phage and Mammalian Origin Cloning: We also have a range of other origins of replication, including the SV40 origin of replication for mammalian cells and the F1 phage origin of replication that can be used to create single stranded DNA.

Plasmid Options for Origins of Replication

Plasmid copy number within individual bacterial cells can vary from very low to very high, and is regulated mainly by the nature of the Origin of Replication used. The origin of replication that is used most frequently in Oxford Genetics plasmids iscalled pUC. It is derived from pBR322 but it contains mutations that remove all constraints on plasmid replication. The bacteria switch from having 15-20 plasmid copies per cell with pBR322 and instead produce up to 700 with pUC. This allows production of large quantities of DNA for research purposes. Other origins of replication featured in Oxford Genetics plasmids include:

• p15A origin (typically 10 copies per cell)
• SC101 (derived from Salmonella, typically 5 copies per cell)
• pBR322 (typically 15-20 copies per cell)
• p15A (typically 10 copies per cell)
• pSC101 (typically 5 copies per cell)

Medium- or low-copy plasmids are often used to minimise unwanted non-specific transgene expression that can occur from high-copy plasmids. Low-copy plasmids produce fewer gene copies, which minimizes background levels and maintains tight regulation of expression. In addition, low copy number origins are particularly useful for large and very large plasmids as this minimises stress to cells.

Plasmids Containing SV40 Origin of Replication

Including the SV40 origin of replicaiton, together with regular bacterial origin, allows propagation of plasmids within mammalian cells expressing the SV40 Large T antigen. This can increase the number of plasmids per cell and thereby increase transgene expression and protein production.

Multiple Cloning Sites / Polylinkers Options

We currently have five alternative multiple cloning sites (polylinkers) in our plasmids. However, most of our plasmids contain the same MCS which is found in all of our plasmids exlcuding the alternatives (pUC19, pUC18, pGem) below.

Most SnapFast plasmids (e.g. pSF-CMV-Amp) (found in almost all SnapFast plasmids except those with alternate multiple cloning sites shown below)

pSF-CMV-pUC19 - The SnapFast Plasmid with the multiple cloning site from the commonly used pUC19 plasmid.

pSF-CMV-pUC18 - The SnapFast Plasmid with the multiple cloning site from the commonly used pUC18 plasmid.

pSF-CMV-pGEM - The SnapFast Plasmid with the multiple cloning site from the commonly used pGem plasmid.

Promoters

We have a wide range of promoters for expression in different biological systems. We also offer vectors that allow you to make your own promoters. All of these promoter options are available flanked by Bgl II restriction sites immediately upstream of the main multiple cloning site (MCS). However, others are also available in other configurations. Therefore, the three positions in our plasmid in which you will find a promoter are as follows:

• Flanked by two Bgl II restriction sites upstream of the main MCS. All promoters are available in this position.
• Flanked by ClaI and BamHI restriction sites. This is currently only available for the PGK promoter for expression in mammalian
• Flanked by AscI restriction site. This is currently only available for the Human Ubiquitin promoter and the Rous Sarcoma virus (RSV) promoter.

Transcriptional Terminators

We have produced two different types of plasmids containing industry standard transcription termination signals. These are:

• Plasmids containing three transcriptional terminators all positioned downstream of the main multiple cloning site (MCS), including the SV40 PolyA signal, RrnG bacterial terminator and the T7 polymerase hair-pin terminator. This single terminator region provides flexibility when exchanging between systems.
• Plasmids containing a single terminator downstream of the MCS.