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Let's Get Started on Your Plasmid DNA Cloning Construct
Our cloning team specializes in creating, modifying, amplifying, and designing plasmid DNA that aligns to specific research objectives. To produce the research grade plasmid DNA researchers need, our team has assembled a comprehensive collection of shuttle vectors, promoters, open reading frames (ORFs), tags, reporters, and inducible systems. The tables below provide more details.
You can begin production by submitting your project details on our request a quote form.
Not sure where to start? No worries. Begin with our step-by-step process to determine the key components of your plasmid construct. If more refinement is needed, our cloning team can provide additional guidance to ensure your project correctly fits your research application.
Step 1: Choose a Backbone
When designing plasmid DNA constructs, choosing a backbone with the appropriate size and capacity is important. Larger backbones can accommodate more genetic material, including larger genes or additional elements like promoters, tags, and reporters. However larger plasmids (i.e. > 18 kb) can be more prone to recombination, leading to instability. BACs and YACs are designed for stable maintenance in host cells, while fosmids and PACs improve stability over cosmids.
Our cloning team offers a variety of backbones each of which presents a unique set of advantages. The list below outlines the differences between backbones so you can select the most appropriate one for your project.
1. Plasmid Vectors for Gene Therapy (e.g., Lentiviral, Adenoviral)
- Capacity: 8-10 kb (Lentiviral), up to 36 kb (Adenoviral)
- Description: Some viral vector backbones used for gene therapy, such as adenoviral vectors, allow for large DNA inserts. While not as large as BACs or cosmids, these viral vectors are large for their type and are used in the delivery of gene therapy constructs.
- Usage: Primarily for gene therapy applications, stable transgene expression in dividing cells, or transient expression.
2. Bacterial Artificial Chromosome (BAC)
- Capacity: 100-300 kb
- Description: BACs are large plasmid backbones designed for cloning very large DNA fragments. They are typically used for constructing genomic libraries or working with very large genes.
- Usage: Often used in genome mapping, complex genetic studies, or generating transgenic animals.
3. Cosmids
- Capacity: 35-45 kb
- Description: Cosmids combine features of plasmids and bacteriophages, allowing for larger inserts than typical plasmids. They have both plasmid elements (such as antibiotic resistance) and cos sequences that allow them to be packaged into bacteriophage capsids for delivery.
- Usage: Common in gene therapy and large-scale cloning projects where higher packaging capacity is required.
4. Yeast Artificial Chromosome (YAC)
- Capacity: 200-1,000 kb
- Description: YACs are used for cloning extremely large DNA fragments and are built from yeast DNA. They allow for the cloning of very large segments of genetic material, which can be stably maintained in yeast cells.
- Usage: Primarily in studying large gene clusters or large-scale genomic projects.
5. P1-derived Artificial Chromosome (PAC)
- Capacity: 100-150 kb
- Description: PACs are similar to BACs but are derived from P1 bacteriophage elements. They are used to clone medium to large DNA fragments.
- Usage: Suitable for large-scale genomic studies and gene therapy applications where larger constructs are needed.
6. Fosmids
- Capacity: 35-40 kb
- Description: Fosmids are similar to cosmids but with a more stable plasmid backbone derived from the F-factor of E. Coli. They are more stable and less prone to recombination than cosmids.
- Usage: Ideal for genomic library construction and gene cloning applications where stability of larger constructs is important.
Step 2: Choose a Promoter
The plasmid DNA construct’s promoter determines where the gene’s transcription is initiated and when/how much of the gene is expressed. If you are working on a project with size constraints or need a strong promoter, choosing the right one from these commonly used examples will depend on your specific research goals.
The list below outlines each promoter’s molecular size, derivation, and typical application.
1. Cytomegalovirus (CMV) Promoter
- Size: ~600 bp
- Description: The CMV promoter is one of the most commonly used strong promoters in mammalian cell expression systems. It’s derived from the human cytomegalovirus and drives high levels of constitutive expression in a broad range of cell types.
- Usage: Widely used in transient and stable gene expression studies, gene therapy, and vaccine development.
2. Simian Virus 40 (SV40) Promoter
- Size: ~200 bp
- Description: Derived from the SV40 virus, this promoter is relatively small compared to others but drives robust expression in a wide range of mammalian cells.
- Usage: Commonly used in plasmids for gene expression in mammalian cells, particularly in conjunction with SV40 enhancer sequences for boosting transcription.
3. Elongation Factor 1-alpha (EF1α) Promoter
- Size: ~1.2 kb
- Description: The EF1α promoter is a strong, constitutive promoter that drives high levels of gene expression in a variety of mammalian cells. It’s a large promoter with a broad activity range.
- Usage: Popular for stable gene expression, especially in gene therapy, cell line generation, and CRISPR-based studies.
4. Ubiquitin C (UBC) Promoter
- Size: ~1.1 kb
- Description: The UBC promoter is derived from the human ubiquitin C gene and drives constitutive gene expression in many cell types. It’s a large promoter that works in a variety of tissues.
- Usage: Frequently used for long-term gene expression in both dividing and non-dividing cells, ideal for gene therapy, lentiviral vectors, and transgenic models.
5. CAG Promoter (CMV early enhancer/chicken β-actin hybrid promoter)
- Size: ~1.6 kb
- Description: The CAG promoter is a hybrid promoter that combines elements from the CMV enhancer and the chicken β-actin promoter, making it one of the strongest and largest constitutive promoters available for mammalian cells.
- Usage: Frequently used in transgenic animals, gene therapy, and vaccine development due to its high expression levels in a wide range of tissues.
6. Human β-Actin (ACTB) Promoter
- Size: ~1.4 kb
- Description: Derived from the human β-actin gene, this promoter drives robust expression in many cell types. It’s often used for constitutive expression of transgenes in a wide variety of tissues.
- Usage: Popular in transgenic research, gene therapy, and when tissue-specific expression isn’t necessary.
7. Muscle Creatine Kinase (MCK) Promoter
- Size: ~1.4 kb
- Description: The MCK promoter drives gene expression specifically in muscle tissues. It’s a larger promoter with tissue-specific applications.
- Usage: Commonly used for gene expression studies in skeletal and cardiac muscle tissue, particularly in models of muscular disease or muscle-targeted gene therapy.
8. Neuron-Specific Enolase (NSE) Promoter
- Size: ~1.3 kb
- Description: The NSE promoter is used to drive gene expression in neurons. It’s relatively large for a tissue-specific promoter and is widely used in neuroscience research.
- Usage: Commonly used in gene expression studies focused on neurons, neurodegenerative diseases, or neuronal transgenic models.
9. Alpha-Myosin Heavy Chain (αMHC) Promoter
- Size: ~4.8 kb
- Description: One of the largest promoters used in plasmid constructs, the αMHC promoter drives gene expression specifically in cardiac tissue.
- Usage: Primarily used in research related to heart function, cardiac disease models, and gene therapy targeting the heart.
10. Tetracycline (Tet)-Responsive Element Promoter (TRE)
- Size: ~1.4 kb (including regulatory regions)
- Description: The TRE promoter is part of the Tet-on/Tet-off inducible system, where gene expression can be tightly controlled by the presence or absence of tetracycline or its analogs.
- Usage: Frequently used in inducible gene expression systems for research that requires temporal control over gene activation.
11. PGK (Phosphoglycerate Kinase) Promoter
- Size: ~500 bp
- Description: Constitutive promoter driving moderate expression in many cell types.
- Usage: Frequently used in viral vectors, transgene expression, and gene therapy.
12. RSV (Rous Sarcoma Virus) Promoter
- Size: ~300 bp
- Description: Strong viral promoter that drives high-level constitutive gene expression.
- Usage: Commonly used in gene therapy vectors and transient gene expression systems.
13. hTERT (Human Telomerase Reverse Transcriptase) Promoter
- Size: ~300 bp
- Description: Promoter active in cancer and immortalized cells, derived from the telomerase gene.
- Usage: Used in cancer research and gene therapy to target immortalized cells.
15. ALB (Albumin) Promoter
- Size: ~2.3 kb
- Description: Liver-specific promoter driving high expression in hepatocytes.
- Usage: Commonly used in liver-targeted gene therapy and research focused on liver function.
16. PDX1 (Pancreatic and Duodenal Homeobox 1) Promoter
- Size: ~850 bp
- Description: Promoter driving expression in pancreatic cells, especially β-cells.
- Usage: Frequently used in diabetes research and pancreas-targeted gene therapy.
17. HSP70 (Heat Shock Protein 70) Promoter
- Size: ~1 kb
- Description: Inducible promoter activated under stress conditions like heat shock.
- Usage: Utilized in research requiring stress-responsive gene expression.
18. LCK Promoter
- Size: ~1.1 kb
- Description: T-cell-specific promoter driving expression in immune cells.
- Usage: Widely used in immunology research for studying T-cell function.
19. GFAP (Glial Fibrillary Acidic Protein) Promoter
- Size: ~2 kb
- Description: Astrocyte-specific promoter for gene expression in glial cells.
- Usage: Commonly used in neuroscience research to target astrocytes.
20. TBG (Thyroxine Binding Globulin) Promoter
- Size: ~460 bp
- Description: Liver-specific promoter often used for high expression in liver cells.
- Usage: Applied in liver-targeted gene therapy and hepatocyte-specific studies.
21. MHC Class II Promoter
- Size: ~900 bp
- Description: Promoter driving gene expression in antigen-presenting cells.
- Usage: Frequently used in immunology and vaccine development.
22. SP-C (Surfactant Protein C) Promoter
- Size: ~400 bp
- Description: Lung-specific promoter used for targeting respiratory epithelial cells.
- Usage: Commonly used in research on lung function and respiratory diseases.
23. Rosa26 Promoter
- Size: ~1.4 kb
- Description: Ubiquitous promoter used in mice for stable gene expression.
- Usage: Frequently used in transgenic mouse models for constitutive expression.
24. Thy1 Promoter
- Size: ~1.2 kb
- Description: Neuron-specific promoter targeting expression in neurons.
- Usage: Widely used in neuroscience for gene expression in the nervous system.
25. Synapsin (Syn) Promoter
- Size: ~500 bp
- Description: Neuron-specific promoter driving expression in mature neurons.
- Usage: Used for studies on neuronal activity and function.
26. c-fos Promoter
- Size: ~1.7 kb
- Description: Activity-dependent promoter activated by neuronal activity.
- Usage: Used in neuroscience to study brain activity and neural circuits.
27. LPL (Lipoprotein Lipase) Promoter
- Size: ~1 kb
- Description: Promoter driving expression in adipocytes.
- Usage: Used in neuroscience to study brain activity and neural circuits.
28. Insulin Promoter
- Size: ~400 bp
- Description: Pancreatic β-cell-specific promoter driving expression in insulin-producing cells.
- Usage: Commonly used in diabetes and endocrinology research.
29. TetO (Tetracycline Operator) Promoter
- Size: ~19 bp (as an operator element)
- Description: Regulatory element used with Tet-repressor for controlling gene expression.
- Usage: Applied in inducible expression systems (Tet-On/Tet-Off).
30. CRP (C-Reactive Protein) Promoter
- Size: ~600 bp
- Description: Liver-specific promoter involved in the immune response.
- Usage: Used in inflammation and immune system research.
31. CK (Creatine Kinase) Promoter
- Size: ~728 bp (3xCK variant)
- Description: Muscle-specific promoter driving expression in skeletal and cardiac muscle.
- Usage: Applied in muscle biology, including studies on muscular disorders.
32. Villin Promoter
- Size: ~1.1 kb
- Description: Promoter driving gene expression in the epithelial cells of the intestine.
- Usage: Used in gastrointestinal research and transgenic models targeting the gut.
33. Peripherin Promoter
- Size: ~400 bp
- Description: Neuron-specific promoter targeting peripheral neurons.
- Usage: Commonly used in studies on peripheral nerve function.
34. Oct4 (POU5F1) Promoter
- Size: ~600 bp
- Description: Pluripotency-specific promoter active in embryonic stem cells.
- Usage: Used in stem cell research and studies on pluripotency.
35. NANOG Promoter
- Size: ~600 bp
- Description: Pluripotency-specific promoter active in embryonic stem cells.
- Usage: Used in stem cell research and studies on pluripotency.
36. EGR1 (Early Growth Response 1) Promoter
- Size: ~1 kb
- Description: Inducible promoter activated by growth factors and stress stimuli.
- Usage: Used in gene expression studies related to cell growth and development.
37. ANF (Atrial Natriuretic Factor) Promoter
- Size: ~300 bp
- Description: Cardiac-specific promoter driving expression in the heart.
- Usage: Applied in cardiovascular research and heart disease models.
39. SOX2 Promoter
- Size: ~600 bp
- Description: Promoter active in neural stem cells and progenitor cells.
- Usage: Applied in neural stem cell research and regenerative medicine.
40. APOE (Apolipoprotein E) Promoter
- Size: ~800 bp
- Description: Liver-specific promoter involved in lipid metabolism.
- Usage: Used in research on neurodegenerative diseases, lipid metabolism, and atherosclerosis.
Step 3: Choose a Shuttle Vector
A plasmid DNA construct’s shuttle vector allows for the transfer of genetic material between two different biological systems such as between bacteria and eukaryotic cells.
Shuttle vectors ensure dual host compatibility, contain selectable markers for both host systems such as antibiotic resistance genes for bacterial hosts and other selection markers for eukaryotic cells, enable the cloning of a gene of interest in one host and then its expression or study in another, and facilitate the transfer of plasmid DNA between hosts.
1. pUC Series (pUC19, pUC18)
- Size: ~2.7 kb
- Host organisms: E. coli and yeast
- Features: High-copy-number plasmids with multiple cloning sites (MCS) and ampicillin resistance. These vectors are widely used for cloning in E. coli before transferring constructs to other hosts.
2. pGEM Series
- Size: ~3.0 kb
- Host organisms: E. coli and yeast
- Features: Contains T7 and SP6 RNA polymerase promoters for in vitro transcription. Common for subcloning and blue/white screening in E. coli.
3. pYES2
- Size: ~5.9 kb
- Host organisms: E. coli and Saccharomyces cerevisiae (yeast)
- Features: Designed for expression in yeast, with a galactose-inducible promoter (GAL1) and URA3 selection marker for yeast.
4. pBR322
- Size: ~4.4 kb
- Host organisms: E. coli and yeast
- Features: One of the oldest and most widely used vectors, containing tetracycline and ampicillin resistance genes. Popular for basic cloning work and shuttle vector applications.
5. pRS Series
- Size: ~5-7 kb (depending on the variant)
- Host organisms: E. coli and S. cerevisiae (yeast)
- Features: These vectors offer yeast selectable markers such as HIS3, TRP1, LEU2, and URA3, and are often used in yeast genetics studies.
6. pCMV Vector Series
- Size: ~4-6 kb (depending on the variant)
- Host organisms: E. coli and mammalian cells
- Features: Designed for high-level expression in mammalian cells, using the cytomegalovirus (CMV) promoter. Often includes antibiotic resistance genes for selection in E. coli (ampicillin, kanamycin) and mammalian cells (neomycin, hygromycin).
7. pBK-CMV
- Size: ~4.9 kb
- Host organisms: E. coli and mammalian cells
- Features: Contains a hybrid SV40-CMV promoter for mammalian expression and kanamycin resistance for selection in E. coli.
8. pBluescript (pBS)
- Size: ~3.0 kb
- Host organisms: E. coli and eukaryotes
- Features: A common high-copy-number cloning vector with a blue/white screening system. It has promoters for both T3 and T7 RNA polymerase, allowing for versatile applications.
9. pET Series
- Size: ~5.3-5.6 kb (depending on the variant)
- Host organisms: E. coli and eukaryotes
- Features: Primarily used for high-level protein expression in E. coli, with T7 promoter-driven expression. Some variants are engineered to function in eukaryotic cells as well.
10. pREP Series
- Size: ~5-6 kb (depending on the variant)
- Host organisms: E. coli and eukaryotes
- Features: Vectors such as pREP1 are used for fission yeast (Schizosaccharomyces pombe) transformation, with nmt1 promoters for regulated expression.
11. pGEX Series
- Size: ~4.9-5.1 kb
- Host organisms: E. coli and mammalian cells
- Features: Contains glutathione S-transferase (GST) fusion system for expressing and purifying tagged proteins. Suitable for protein expression in E. coli and some mammalian cell applications.
12. pAAV Series
- Size: ~4-6 kb (depending on the variant)
- Host organisms: E. coli and mammalian cells
- Features: AAV (adeno-associated virus) shuttle vectors used for gene delivery in mammalian systems. Widely employed for gene therapy and functional studies in mammalian cells.
13. pLenti Series
- Size: ~9-11 kb (depending on the variant)
- Host organisms: E. coli and mammalian cells
- Features: Lentiviral-based shuttle vectors that allow stable integration of genetic material into mammalian genomes. Commonly used in gene therapy and transduction experiments.
14. pRSFDuet Series
- Size: ~5.3 kb
- Host organisms: E. coli and eukaryotes
- Features: Designed for co-expression of multiple genes in E. coli, using T7 RNA polymerase promoters. Also used as shuttle vectors for some eukaryotic systems.
15. pDual GC
- Size: ~7-8 kb
- Host organisms: E. coli and mammalian cells
- Features: This vector contains two distinct promoters for co-expressing genes in both E. coli and mammalian cells, often used for dual-function studies.
16. pZeoSV
- Size: ~3.5 kb
- Host organisms: E. coli and mammalian cells
- Features: Contains the SV40 early promoter for expression in mammalian cells and Zeocin resistance for selection in both E. coli and mammalian cells.
17. pTRE-Tight
- Size: ~3.6 kb
- Host organisms: E. coli and mammalian cells
- Features: Utilizes the Tet-On system for tightly regulated expression of genes in mammalian cells, with tetracycline or doxycycline control.
18. pAdTrack
- Size: ~7.9 kb
- Host organisms: E. coli and mammalian cells
- Features: An adenoviral shuttle vector, often used for generating recombinant adenoviruses for gene expression in mammalian cells.
19. pWB980
- Size: ~5-6 kb (depending on the variant)
- Host organisms: E. coli and Bacillus subtilis
- Features: Used for cloning and expression in E. coli and B. subtilis, a model Gram-positive bacterium.
20. pPICZ Series
- Size: ~3.6 kb
- Host organisms: E. coli and Bacillus subtilis
- Features: Contains AOX1 promoter for methanol-inducible expression in P. pastoris. Includes Zeocin resistance for selection in both E. coli and yeast.
21. pESC Series
- Size: ~7-8 kb (depending on the variant)
- Host organisms: E. coli and Saccharomyces cerevisiae (yeast)
- Features: Designed for inducible expression in yeast using the GAL1 and GAL10 promoters. Common in yeast genetics and functional studies.
22. pCH Series
- Size: ~5-7 kb (depending on the variant)
- Host organisms: E. coli and Saccharomyces cerevisiae
- Features: Shuttle vector for cloning in E. coli and yeast, with yeast-specific selectable markers and a CEN/ARS origin for stable propagation in yeast.
23. pDEST Series
- Size: ~7-9 kb (depending on the variant)
- Host organisms: E. coli and mammalian cells
- Features: Utilizes the Gateway cloning system for efficient cloning and expression of genes in both bacterial and mammalian systems.
24. pBP Series
- Size: ~7-8 kb
- Host organisms:E. coli and Schizosaccharomyces pombe (fission yeast)
- Features: Vectors designed for regulated gene expression in fission yeast, with nmt1 promoters for controlled expression.
25. pYAC Series (Yeast Artificial Chromosome)
- Size: Varies greatly, up to 1,000 kb or more (due to its large DNA carrying capacity)
- Host organisms: E. coli and yeast
- Features: Large-capacity vectors (up to several hundred kilobases) used to clone large fragments of DNA in yeast. Essential for studying large genomic regions.
26. pBI121
- Size: ~14.7 kb
- Host organisms: E. coli and plants
- Features: Contains the CaMV 35S promoter for constitutive expression in plants. Often used for Agrobacterium-mediated transformation of plant species.
27. pGreen Series
- Size: ~5.9 kb
- Host organisms: E. coli and plants
- Features: Designed for transformation of plants via Agrobacterium tumefaciens, with selectable markers for both E. coli and plant systems.
28. pCAMBIA Series
- Size: ~9-11 kb (depending on the variant)
- Host organisms: E. coli and plants
- Features: Contains a variety of plant selection markers and promoters (such as CaMV 35S) for transformation and gene expression in plants.
Step 4: Choose an Open Reading Frame (ORF)
The ORF represents the sequence of DNA that can be translated into a protein, making it essential for protein expression studies, functional assays, or production of the protein of interest. The below list of ORFs are some of the options to be cloned into your plasmid DNA construct.
1. GFP (Green Fluorescent Protein)
- Size: ~720 bp
- Description: Fluorescent protein that emits green light when excited with blue or UV light.
- Origin: E. coli and plants
2. RFP (Red Fluorescent Protein)
- Size: ~675 bp
- Description: Fluorescent protein that emits red light, used in multi-color fluorescence studies.
- Origin: Sea anemone Discosoma.
3. Luciferase
- Size: ~675 bp
- Description: An enzyme that catalyzes a light-emitting reaction in the presence of luciferin and oxygen.
- Origin: Firefly Photinus pyralis.
4. Beta-galactosidase (lacZ)
- Size: ~3,072 bp
- Description: Enzyme that breaks down lactose, used in blue/white screening.
- Origin: E. Coli.
5. Beta-lactamase
- Size: ~861 bp
- Description: Provides resistance to beta-lactam antibiotics like ampicillin.
- Origin: E. coli.
6. Chloramphenicol Acetyltransferase (CAT)
- Size: ~666 bp
- Description: Provides resistance to the antibiotic chloramphenicol.
- Origin: E. coli.
7. EGFP (Enhanced Green Fluorescent Protein)
- Size: ~720 bp
- Description: An enhanced variant of GFP with improved brightness and stability.
- Origin: Modified version of Aequorea victoria GFP.
8. mCherry
- Size: ~708 bp
- Description: A monomeric red fluorescent protein, used for imaging in cells and tissues.
- Origin: Engineered from Discosoma RFP.
9. BFP (Blue Fluorescent Protein)
- Size: ~720 bp
- Description: Fluorescent protein that emits blue light, often used in multi-color assays.
- Origin: Engineered from GFP (Aequorea victoria).
10. mVenus
- Size: ~717 bp
- Description: A variant of YFP (Yellow Fluorescent Protein) with increased brightness and faster maturation.
- Origin: Engineered from Aequorea victoria GFP.
11. p53
- Size: ~1,179 bp
- Description: Tumor suppressor protein involved in cell cycle regulation and apoptosis.
- Origin: Human (Homo sapiens).
12. GUS (Beta-Glucuronidase)
- Size: ~1,809 bp
- Description: Used as a reporter enzyme in plants for histochemical and fluorometric detection.
- Origin: E. Coli.
13. Cerulean
- Size: ~720 bp
- Description: A bright cyan fluorescent protein used in FRET and multi-color fluorescence studies.
- Origin: Derived from GFP (Aequorea victoria).
14. Sulfotransferase
- Size: ~750–1,200 bp (varies by species)
- Description: Enzyme involved in post-translational modification of proteins through sulfation.
- Origin: Eukaryotic cells, including human and animal sources.
15. Nluc (NanoLuciferase)
- Size: ~513 bp
- Description: A small and bright luciferase used for sensitive bioluminescent assays.
- Origin: Engineered from Oplophorus gracilirostris (a type of shrimp).
16. mOrange
- Size: ~687 bp
- Description: A bright orange fluorescent protein used for multi-color fluorescence imaging.
- Origin: Derived from Discosoma RFP.
17. mRuby
- Size: ~708 bp
- Description: A red fluorescent protein known for high photostability and brightness.
- Origin: Engineered from Entacmaea quadricolor.
18. eCFP (Enhanced Cyan Fluorescent Protein)
- Size: ~717 bp
- Description: A variant of cyan fluorescent protein (CFP) used in multi-color assays.
- Origin: Derived from GFP (Aequorea victoria).
19. BFP2
- Size: ~717 bp
- Description: Improved version of BFP with enhanced brightness and stability.
- Origin: Engineered from GFP (Aequorea victoria).
20. mTagBFP2
- Size: ~705 bp
- Description: A more photostable and bright version of blue fluorescent protein.
- Origin: Engineered from Entacmaea quadricolor.
21. P2A Peptide
- Size: ~57 bp
- Description: Self-cleaving peptide that allows for the production of two separate proteins from one transcript.
- Origin: Foot-and-mouth disease virus (FMDV).
22. Cre Recombinase
- Size: ~1,029 bp
- Description: Enzyme used for site-specific recombination in genetic engineering (e.g., Cre-Lox system).
- Origin: Bacteriophage P1.
23. Fluc (Firefly Luciferase)
- Size: ~1,650 bp
- Description: Luciferase that produces bioluminescence, used in reporter assays.
- Origin: Firefly Photinus pyralis.
23. Fluc (Firefly Luciferase)
- Size: ~1,650 bp
- Description: Luciferase that produces bioluminescence, used in reporter assays.
- Origin: Firefly Photinus pyralis.
24. Rluc (Renilla Luciferase)
- Size: ~933 bp
- Description: A luciferase enzyme used in bioluminescence assays, typically for dual-luciferase systems.
- Origin: Sea pansy Renilla reniformis.
Optional Step 5: Add a Tag, Reporter, Inducible Reporter, and or Inducible System
Our molecular cloning team offers a variety of tags, reporters, inducible reporter, and inducible systems that can be cloned on to your desired plasmid DNA construct.
Tags: Tags in plasmid DNA constructs streamline the purification, detection, and functional analysis of the expressed proteins in plasmid-based studies. The role of a tag on a plasmid DNA construct is to facilitate various experimental processes including protein purification, protein detection, protein localization, and improving protein solubility. The below list of tags are some of the available options for molecular cloning productions.
1. His-Tag
- Size: ~18–30 bp (typically 6x histidine codons)
- Description: A polyhistidine tag used for affinity purification via nickel chromatography.
- Origin: Synthetic, widely used in recombinant protein expression systems.
2. FLAG Tag
- Size: ~24 bp
- Description: A short peptide tag (DYKDDDDK) used for detection and purification.
- Origin: Synthetic, designed for high affinity to anti-FLAG antibodies.
3. HA Tag (Hemagglutinin Tag)
- Size: ~27 bp
- Description: A short peptide tag (YPYDVPDYA) used for detection, derived from the human influenza virus hemagglutinin protein.
- Origin: Influenza A virus.
4. MYC Tag
- Size: ~30 bp
- Description: A short peptide tag (EQKLISEEDL) derived from the c-Myc transcription factor, used for detection and protein interaction studies.
- Origin: Human c-Myc protein.
5. Strep-Tag
- Size: ~24 bp
- Description: A short peptide tag (WSHPQFEK) used for affinity purification via streptavidin.
- Origin: Synthetic, developed for high affinity to streptavidin.
6. TAP Tag (Tandem Affinity Purification)
- Size: ~252 bp
- Description: A dual-tag system combining protein A and calmodulin-binding peptide (CBP) for high-purity protein purification.
- Origin: Synthetic, developed for sequential purification.
7. MBP Tag (Maltose-Binding Protein)
- Size: ~1,200 bp
- Description: A large protein tag that improves protein solubility and aids in purification through binding to amylose resin.
- Origin: E. Coli maltose-binding protein.
8. SUMO Tag
- Size: ~300 bp
- Description: A protein tag that enhances solubility and stability, often cleavable by SUMO protease.
- Origin: Derived from Small Ubiquitin-like Modifier (SUMO) proteins in eukaryotes.
9. GST Tag (Glutathione-S-Transferase)
- Size: ~660 bp
- Description: A protein tag used for purification by binding to glutathione resin, and for studying protein-protein interactions.
- Origin: Schistosoma japonicum.
10. FLAG-Myc Tag
- Size: ~54 bp
- Description: A combined dual tag (DYKDDDDK-EQKLISEEDL) for enhanced detection and purification.
- Origin: Synthetic combination of FLAG and Myc tags.
11. HA-Tag (Hemagglutinin Tag)
- Size: ~27 bp
- Description: A peptide tag (YPYDVPDYA) used for detection and interaction studies.
- Origin: Influenza A virus.
12. C-Myc Tag
- Size: ~30 bp
- Description: A short tag (EQKLISEEDL) used for detection and analysis of protein interactions.
- Origin: Derived from human c-Myc transcription factor.
13. Biotinylation Tag
- Size: ~66 bp
- Description: A short peptide tag that allows for enzymatic biotinylation, facilitating detection and purification using streptavidin.
- Origin: Synthetic.
14. TEV Protease Site
- Size: ~21 bp
- Description: A cleavage site for Tobacco Etch Virus (TEV) protease (ENLYFQG), allowing the removal of tags post-purification.
- Origin: Tobacco Etch Virus.
15. V5 Tag
- Size: ~42 bp
- Description: A peptide tag (GKPIPNPLLGLDST) used for detection and purification, derived from the P and V proteins of simian virus 5 (SV5).
- Origin: Simian Virus 5 (SV5).
16. S-tag
- Size: ~27 bp
- Description: A small peptide tag (KETAAAKFERQHMDS) used for detection and purification, with high affinity to S-protein.
- Origin: Derived from ribonuclease A (Bovine pancreas).
17. Myc-DDK Tag
- Size: ~54 bp
- Description: A dual peptide tag (EQKLISEEDL-DYKDDDDK) combining Myc and DDK (FLAG) for detection and purification.
- Origin: Synthetic.
18. GFP-Tag
- Size: ~720 bp
- Description: A GFP fusion tag for visualization of protein localization and real-time expression in cells.
- Origin: Aequorea victoria.
19. HIS-MBP Tag
- Size: ~1,218 bp
- Description: A dual tag combining a His-tag and Maltose-Binding Protein (MBP) for enhanced solubility and affinity purification.
- Origin: E. Coli.
21. N-terminal His-Tag
- Size: ~18–30 bp
- Description: A polyhistidine tag placed at the N-terminus of the protein for affinity purification.
- Origin: Synthetic.
22. C-terminal His-Tag
- Size: ~45 bp
- Description: A short peptide tag (GLNDIFEAQKIEWHE) that can be biotinylated enzymatically for detection and purification.
- Origin: Synthetic, developed for biotin ligase recognition.
23. S-Tag
- Size: ~27 bp
- Description: A small peptide tag (KETAAAKFERQHMDS) used for detection and purification.
- Origin: Derived from ribonuclease A (Bovine pancreas).
24. 6xHis Tag
- Size: ~18 bp
- Description: A short tag of six histidine residues used for affinity purification via metal ions like nickel or cobalt. It allows easy detection and purification of recombinant proteins.
- Origin: Derived from ribonuclease A (Bovine pancreas).
Reporters: Reporters help validate whether genetic elements in a plasmid are functioning as intended. is a gene that encodes for a detectable protein, which allows researchers to monitor the activity of the plasmid in a cell or organism. The role of a reporter gene is primarily to provide a visual or measurable readout that indicates the success of transfection, gene expression, or regulatory activity within the plasmid construct.
The below list of reporters are some of the available options for molecular cloning productions.
1. eGFP (Enhanced Green Fluorescent Protein)
- Size: ~720 bp
- Description: A modified version of the original GFP from Aequorea victoria jellyfish. Enhanced for brighter fluorescence and faster folding.
- Origin: Aequorea victoria jellyfish, with mutations to improve brightness and reduce issues with folding in mammalian cells.
Learn more about GFP lentivirus and why this protein is so prevalent in molecular biology research.
2. mCherry
- Size: ~711 bp
- Description: A red fluorescent protein that matures quickly and is relatively photostable. Emission peak at 610 nm.
- Origin:Derived from DsRed, a red fluorescent protein from Discosoma sea anemones, engineered for faster maturation and monomeric properties.
3. mStrawberry
- Size: ~720 bp
- Description: A red-orange fluorescent protein with an emission peak around 581 nm. It’s brighter than many red fluorescent proteins.
- Origin: Also derived from DsRed of Discosoma, engineered for faster maturation and brighter fluorescence.
4. RFP (Red Fluorescent Protein)
- Size: ~700 bp
- Description: A broad term that can refer to several different red fluorescent proteins, typically derived from DsRed. Used for red-shifted fluorescence emission.
- Origin: Originally isolated from Discosoma species (sea anemones).
5. LacZ (β-galactosidase)
- Size: ~3,000 bp
- Description: Encodes an enzyme that catalyzes the hydrolysis of lactose. Used with substrates like X-gal, producing a blue color when expressed.
- Origin: Derived from E. Coli as part of the lac operon.
6. Luciferase (Firefly Luciferase)
- Size: ~1,650 bp
- Description: Catalyzes the oxidation of luciferin, producing light. Widely used in bioluminescence assays.
- Origin: Derived from Photinus pyralis (North American firefly).
7. mOrange
- Size: ~700 bp
- Description: A bright monomeric orange fluorescent protein with an emission peak at 562 nm.
- Origin: Engineered from Discosoma sp.
8. mRFP1 (monomeric Red Fluorescent Protein 1)
- Size: ~700 bp
- Description: First true monomeric red fluorescent protein, optimized for faster folding and expression.
- Origin: Derived from Discosoma (sea anemones).
9. EYFP (Enhanced Yellow Fluorescent Protein)
- Size: ~717 bp
- Description: A variant of GFP optimized for yellow emission (~527 nm), with improved brightness and photostability.
- Origin: Derived from Aequorea victoria, with mutations for yellow fluorescence.
10. CFP (Cyan Fluorescent Protein)
- Size: ~720 bp
- Description: A GFP variant optimized for cyan emission (~476 nm), used in FRET (Förster resonance energy transfer) experiments.
- Origin: Engineered from Aequorea victoria GFP.
11. DsRed (Discosoma Red Fluorescent Protein)
- Size: ~700 bp
- Description: A red fluorescent protein with emission around 583 nm, originally oligomeric but later monomerized to create variants like mCherry.
- Origin: Derived from Discosoma (sea anemones).
12. tdTomato
- Size: ~1,400 bp (tandem dimer)
- Description: A tandem dimer fluorescent protein with high brightness and red emission (581 nm).
- Origin: Engineered from DsRed for enhanced brightness.
13. Venus
- Size: ~720 bp
- Description: A variant of yellow fluorescent protein (YFP) with faster maturation and less sensitivity to pH.
- Origin: Derived from GFP.
14. BFP (Blue Fluorescent Protein)
- Size: ~720 bp
- Description: A blue variant of GFP with an emission peak around 447 nm. Used in FRET applications and for multi-color labeling.
- Origin: Engineered from GFP.
15. NanoLuc (Nano Luciferase)
- Size: ~513 bp
- Description: A small luciferase that produces a bright luminescent signal, often used in biosensors and live cell imaging.
- Origin: Derived from a deep-sea shrimp Oplophorus gracilirostris.
16. Gaussia Luciferase
- Size: ~558 bp
- Description: A secreted luciferase from marine copepod with very bright luminescence, used in real-time live-cell assays.
- Origin: Derived from Gaussia princeps.
17. Renilla Luciferase
- Size: ~936 bp
- Description: A luciferase that catalyzes bioluminescence in the presence of coelenterazine, widely used for dual-reporter assays.
- Origin: Derived from Renilla reniformis (sea pansy).
18. GUS (β-Glucuronidase)
- Size: ~1,800 bp
- Description: Encodes an enzyme that cleaves glucuronides, often producing a blue color in histochemical assays.
- Origin: Derived from E. Coli.
19. Peroxidase (HRP, Horseradish Peroxidase)
- Size: ~1,200 bp
- Description: Produces a colorimetric or luminescent signal in the presence of hydrogen peroxide, used in ELISA and western blotting.
- Origin: Derived from Armoracia rusticana (horseradish plant).
20. Gluc (Glucose Oxidase)
- Size: ~4,500 bp
- Description: Produces a measurable signal by oxidizing glucose to produce hydrogen peroxide. Used in biosensor applications.
- Origin: Derived from fungi such as Aspergillus niger.
Inducible Reporters: An inducible reporter gene, like GFP or luciferase, is often used to monitor the success of induction. After molecular cloning the gene onto the plasmid DNA construct, the reporter gene’s expression can be easily measured by fluorescence or luminescence, giving a visual or quantitative indication of the activity of the inducible system. An inducible reporter gene is regulated by an external stimulus (such as a chemical, light, or temperature). Inducible reporters are expressed only when the inducer is present, allowing for precise temporal and environmental control over when the reporter gene is activated.
The below list of inducible reporters are some of the available options for molecular cloning productions.
1. pBAD-GFP
- Size: ~700 bp for GFP, ~1.2 kb for pBAD regulatory elements
- Description: A fusion of the arabinose-inducible pBAD promoter with the GFP reporter gene, allowing visual tracking of expression in response to arabinose.
- Origin: Synthetic construct derived from the E. coli arabinose operon.
2. Luciferase Reporter (Firefly)
- Size: ~1.7 kb
- Description: Encodes the enzyme luciferase, which catalyzes a reaction that produces bioluminescence. Often used as a reporter in inducible systems for real-time tracking of gene expression.
- Origin: Firefly (Photinus pyralis).
3. Luciferase Reporter (Renilla)
- Size: ~900 kb
- Description: Similar to firefly luciferase, Renilla luciferase emits light when its substrate coelenterazine is present. Used as a dual-reporter system.
- Origin: Sea pansy (Renilla reniformis).
4. GFP (Green Fluorescent Protein) Reporter
- Size: ~720 kb
- Description: GFP produces green fluorescence when excited by UV or blue light, making it a versatile reporter in various inducible systems.
- Origin: Jellyfish (Aequorea victoria).
5. mCherry Reporter
- Size: ~711 kb
- Description: A red fluorescent protein variant, commonly used in inducible systems to track gene expression in live cells.
- Origin: Modified from Discosoma (a type of coral).
Inducible Systems: The inducible system controls gene expression by regulating when a target gene is turned on or off in response to specific environmental or chemical signals. On a plasmid DNA construct, an regulates the expression of a specific gene of interest, while an inducible reporter is used to track or confirm that induction is occurring.
The below list of inducible systems are some of the available options for molecular cloning productions.
1. T7 Promoter System
- Size: ~500 bp (base pairs) for the promoter region.
- Description: A strong promoter used in bacteriophage T7 RNA polymerase systems. It is highly specific and requires the presence of T7 RNA polymerase for expression.
- Origin: Bacteriophage T7.
2. Lac Operon (LacI/IPTG) System
- Size: LacI gene (~1 kb), lac operator (~20 bp), and the lacZ gene (reporter) (~3 kb).
- Description: A classic inducible system where gene expression is repressed by LacI and induced by IPTG (isopropyl β-D-1-thiogalactopyranoside). Often coupled with β-galactosidase as a reporter.
- Origin: E. coli.
3. Tet-On/Tet-Off Systems
- Size: ~2.2 kb for TetR and ~200 bp for TetO operator.
- Description: In the Tet-On system, gene expression is induced by the presence of tetracycline or doxycycline. In the Tet-Off system, gene expression is turned off by these molecules. It is tightly controlled and used widely in mammalian cells.
- Origin: Tetracycline resistance operon from bacteria.
4. AraC/PBAD System
- Size: ~900 bp for AraC gene, ~300 bp for PBAD promoter.
- Description: A system that responds to the presence of arabinose to induce gene expression, commonly used in bacterial expression systems.
- Origin: E. coli in arabinose operon.
5. rtTA (Reverse Tetracycline Transactivator)
- Size: ~900 bp.
- Description: This modified version of the Tet-On system works in reverse, where doxycycline activates the transactivator that binds TetO to induce gene expression.
- Origin: Derived from the tetracycline resistance operon with synthetic modifications.
6. CRE/loxP System
- Size: ~1.1 kb for CRE recombinase, loxP sites ~34 bp each.
- Description: An inducible gene editing system where CRE recombinase excises DNA sequences flanked by loxP sites, enabling precise genetic control.
- Origin: Bacteriophage P1.
7. Estrogen Receptor (ER)-based Inducible Systems
- Size: ~1.2 kb for the estrogen receptor (ER) domain.
- Description: Gene expression is induced by the presence of tamoxifen, which binds the modified ER domain. Often used in conditional knockout experiments.
- Origin: Mammalian estrogen receptor.
8. Heat Shock Promoter System (Hsp70)
- Size: ~2 kb for the full promoter region.
- Description: A promoter activated by heat shock (increased temperature), used for inducing gene expression under stress conditions.
- Origin: Heat shock genes from various organisms, including Drosophila melanogaster and E. coli.
9. CRISPR-Cas9 Inducible System
- Size: ~4.2 kb for Cas9, ~100 bp for gRNA.
- Description: An inducible form of the CRISPR-Cas9 system, where gene editing is controlled by inducible promoters to activate Cas9 expression only when needed.
- Origin: Streptococcus pyogenes Cas9 system.
10. pLysS/T7-lacO System
- Size: ~400 bp for the T7 promoter, ~1 kb for lacI gene.
- Description: A dual regulation system that combines T7 and lac operons, where expression is tightly controlled by both IPTG and the host’s lac operon system.
- Origin: Hybrid of E. coli and bacteriophage T7 systems.
Optional Step 6: Package Plasmid DNA Construct into Viral Vector Particles
Our viral vector team works hand-in-hand with our cloning team and can easily package your custom plasmid DNA construct into adenoviral, adeno-associated viral, lentiviral, or retroviral particles. For more information check out our viral vector services:
Plasmid DNA Integrated Vector Sequencing Reports for IND Filing
Integrated Vector Sequencing Reports are a critical component of any Investigational New Drug application submitted to the FDA for gene therapy products. This analysis provides essential verification of the vector’s genetic integrity, identity, and stability. By documenting the complete nucleotide sequence of a therapeutic vector, identifying any potential sequence variants, and confirming genetic stability across manufacturing processes, this report addresses crucial regulatory requirements while demonstrating a commitment to product quality.
Our plasmid DNA team can provide Integrated Vector Sequencing Reports for plasmid DNA vectors, just ask when starting a project.
Molecular Cloning in Biopharmaceutical and Biotechnology Research & Development
Molecular cloning is a versatile technique that allows researchers to create the specific plasmid DNA constructs they need to research and develop cell and gene therapies, study pharmacogenomics, observe disease biomarkers, and develop vaccines.
Precision medicine research and development progresses everyday, and with it, the need for high quality custom plasmid DNA constructs.
Want to learn more about the latest in molecular cloning? Our colleagues at ScienceDirect, the American Society for Biochemistry and Molecular Biology, and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on molecular cloning.
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