GFP Fusion Calculator
Design Your GFP Fusion Protein
About the GFP Fusion Calculator
The GFP Fusion Calculator is a free, accurate, and scientifically validated online tool that instantly designs N-terminal, C-terminal, or internal GFP fusion proteins from any target protein sequence. Widely used in molecular biology, cell biology, and synthetic biology, GFP (Green Fluorescent Protein) fusions allow real-time visualization of protein localization, dynamics, and interactions in living cells.
What is a GFP Fusion Protein?
A GFP fusion protein is created by genetically linking the coding sequence of GFP (or its improved variants) to the gene of your protein of interest. The most common configurations are:
- N-terminal fusion: GFP–Linker–Protein
- C-terminal fusion: Protein–Linker–GFP (most frequently used)
- Internal fusion: Used for domain tracking or split-GFP systems
Importance of GFP Fusion Proteins
GFP and its derivatives revolutionized biological research (Nobel Prize in Chemistry 2008 – Osamu Shimomura, Martin Chalfie, Roger Tsien). Today, fluorescent protein fusions are indispensable for:
- Live-cell imaging and protein localization studies
- Protein–protein interaction studies (FRET, BiFC)
- High-throughput screening and reporter assays
- Tracking protein trafficking, degradation, and expression levels
- CRISPR knock-in tagging strategies
Scientific Foundation & Parameters Used
This GFP Fusion Calculator uses peer-reviewed, experimentally validated data:
- Molecular weights and extinction coefficients from FPbase.org and Tsien Lab publications
- pI calculation via the Bjellqvist scale (EMBOSS/Expasy standard)
- Brightness values relative to EGFP = 100 (Lam et al., Nat Methods 2012)
- Standard flexible (GGGGS)n and rigid linkers from literature (Chen et al., Adv Drug Deliv Rev 2013)
When and Why You Should Use This Tool
Use the GFP Fusion Calculator when you are:
- Designing plasmids for transient transfection or stable cell lines
- Planning CRISPR-based endogenous tagging
- Comparing brightness and folding efficiency between GFP variants
- Optimizing linker length to preserve protein function and fluorescence
- Teaching molecular biology or synthetic biology courses
User Guidelines for Best Results
- Enter only the mature protein sequence (without signal peptides unless desired)
- C-terminal fusions are generally preferred as they less frequently interfere with localization signals
- Use flexible linkers ≥10 aa to avoid steric hindrance
- Superfolder GFP (sfGFP) is recommended for poorly folding or aggregation-prone proteins
- mNeonGreen is currently the brightest monomeric option
This completely free GFP Fusion Calculator is proudly brought to you by Agri Care Hub. For detailed protocols and background, visit GFP Fusion Proteins – LibreTexts.
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