Type IIS restriction enzymes — enzymes that cut a defined distance outside their recognition site — are the engine of scarless Golden Gate and MoClo DNA assembly. This application note maps BioHippo's ten restriction enzymes, including the FuniCut™ fast series that reaches complete digestion in 5–15 minutes on a single universal buffer, to the cloning and biomanufacturing workflows where they fit best.
What are Type IIS restriction enzymes?
Restriction enzymes fall into two practical groups for the bench. Classic Type II enzymes (EcoRI, BamHI, AvaII) recognize a short palindromic sequence and cut within it, leaving a fixed overhang. Type IIS restriction enzymes (BsaI, BbsI, Esp3I/BsmBI, EarI, BspQI/SapI-class) recognize an asymmetric sequence and cut a short, defined distance away from it.
Because the cut site sits outside the recognition sequence, the enzyme site is removed from the final product. That single property enables scarless, directional, one-pot assembly — the basis of Golden Gate cloning, introduced by Engler and colleagues in 2008,[1] and of the hierarchical MoClo (Modular Cloning) standard described by Weber et al. in 2011.[2] These methods are now routine for building multigene constructs in metabolic engineering, plant and mammalian synthetic biology, CRISPR guide-RNA and vector construction, and biosensor design.
Why fast restriction enzymes change the workflow
Traditional restriction digests are a bottleneck: overnight incubations, enzyme-specific buffers, and staggered double digests that stretch a simple cloning step across a full day. The FuniCut™ series of fast restriction enzymes is built to remove that bottleneck.
Each FuniCut™ enzyme is produced by recombinant engineering and optimized for speed. A typical reaction reaches complete digestion in 5–15 minutes at 37 °C — plasmid DNA in ~15 min, PCR products in 15–30 min, and genomic DNA in 30–60 min. Because every enzyme in the series shares one universal 10× FuniCut™ Buffer, you can combine enzymes for double and multiple digestions in a single tube, with no sequential buffer swaps.
One tube, one buffer, one short incubation. Set up the digest, incubate for a coffee break, and load straight to gel — the optional 10× FuniCut™ Color Buffer carries red and yellow tracking dyes so reaction products can be loaded onto agarose gels without a separate loading buffer.
- Rapid digestion — complete cleavage in 5–15 minutes versus the classic 1–16 hour incubation.
- Universal buffer — one reaction buffer across the series enables true one-tube multi-enzyme digests.
- Activity redundancy — high enzyme-activity margin handles excess substrate and difficult templates.
- Direct gel loading — optional Color Buffer with built-in tracking dyes: digest, then load without extra steps.
The 10 restriction enzymes at a glance
BioHippo's restriction enzyme collection pairs eight FuniCut™ fast enzymes with two BspQI options (research- and GMP-grade) for template linearization. Type IIS enzymes cut outside their recognition sequence — ideal for scarless assembly such as Golden Gate and MoClo — while classic Type II enzymes cut within a fixed palindromic site.
| Enzyme | Cat. No. | Type | Recognition / cut site | Activity | Temp | Heat inact. |
|---|---|---|---|---|---|---|
| FuniCut™ EcoRI | BHZ20800115 | Type II | 5'-G↓AATTC-3' | 20 U/µL | 37 °C | 80 °C, 20 min |
| FuniCut™ BamHI | BHZ20800113 | Type II | 5'-G↓GATCC-3' | 20 U/µL | 37 °C | Not heat-inact. |
| FuniCut™ AvaII | BHZ20800118 | Type II | 5'-G↓GWCC-3' | 10 U/µL | 37 °C | 80 °C, 20 min |
| FuniCut™ BsaI | BHZ20800114 | Type IIS | 5'-GGTCTC(N)1↓-3' | 20 U/µL | 37 °C | 80 °C, 20 min |
| FuniCut™ BbsI | BHZ20800079 | Type IIS | 5'-GAAGAC(N)2↓-3' | 20 U/µL | 37 °C | 80 °C, 20 min |
| FuniCut™ EarI | BHZ20800117 | Type IIS | 5'-CTCTTC(N)1↓-3' | 20 U/µL | 37 °C | 80 °C, 20 min |
| FuniCut™ Esp3I (BsmBI) | BHZ20800116 | Type IIS | 5'-CGTCTC(N)1↓-3' | — | 37 °C | Yes |
| FuniCut™ DpnI | BHZ20800014 | Type II | 5'-GA(m6A)↓TC-3' | — | 37 °C | Yes |
| BspQI | BHZ20800119 | Type IIS | 5'-GCTCTTC(N)1↓-3' | 10 U/µL | 50 °C | — |
| BspQI GMP-grade | BHZ20800005 | Type IIS · GMP | 5'-GCTCTTC(N)1↓-3' | 10 U/µL | 50 °C | — |
W = A or T. (N)n indicates cleavage a defined number of bases outside the recognition sequence — the signature of Type IIS enzymes. All FuniCut™ enzymes are supplied with 10× FuniCut™ Buffer and optional 10× FuniCut™ Color Buffer.
Type IIS enzymes for Golden Gate & scarless cloning
Because Type IIS enzymes cut outside their recognition site, the site is removed from the final product, enabling scarless, directional, one-pot assembly — the foundation of Golden Gate and MoClo workflows.
FuniCut™ BsaI — GGTCTC(N)1↓
The workhorse of Golden Gate assembly. Generates four-base overhangs for high-fidelity, multi-fragment one-pot reactions. 20 U/µL. Unaffected by Dam, Dcm, EcoKI, or EcoBI methylation; CpG methylation overlaps the site and blocks cleavage.
FuniCut™ BbsI — GAAGAC(N)2↓
A second orthogonal Golden Gate enzyme (isoschizomers BstV2I, BpiI), ideal for hierarchical/MoClo assemblies alongside BsaI. Unaffected by Dam, Dcm, CpG, EcoKI, and EcoBI methylation.
FuniCut™ Esp3I (BsmBI) — CGTCTC(N)1↓
BsmBI isoschizomer for a second assembly tier in MoClo/Loop workflows. Heat-inactivatable; isoschizomers include BsmBI, Esp16I, Esp23I.
FuniCut™ EarI — CTCTTC(N)1↓
Cuts one base outside a 6-bp recognition site to leave three-base overhangs — useful for custom overhang designs and specialized assembly. 20 U/µL.
BspQI — GCTCTTC(N)1↓
Type IIS enzyme (SapI class) recognizing a 7-bp site and generating three-base overhangs for seamless cloning and plasmid linearization. Reacts at 50 °C; research-use grade.
Type II enzymes for everyday cloning
FuniCut™ EcoRI — G↓AATTC
One of the most widely used enzymes in molecular biology. Generates 5' AATT overhangs for routine subcloning and screening. 20 U/µL, heat-inactivatable.
FuniCut™ BamHI — G↓GATCC
Classic 5' GATC overhang generator for vector and insert preparation. Activity unaffected by Dam, Dcm, CpG, EcoKI, or EcoBI methylation. Purify by phenol-chloroform or column (not heat-inactivatable).
FuniCut™ AvaII — G↓GWCC
Degenerate-site enzyme (W = A/T) for mapping and fragment analysis. Isoschizomers Bme18I, Eco47I. 10 U/µL, heat-inactivatable.
Specialty & GMP: DpnI cleanup and IVT-grade linearization
FuniCut™ DpnI — methylation-dependent
DpnI cleaves only Dam-methylated DNA (GAm6TC), making it the standard tool for removing the methylated parental template after site-directed mutagenesis, and for SNP analysis, Southern blotting, and genotyping. Heat-inactivatable; not Type IIS.
BspQI GMP-grade — for regulated workflows
The same GCTCTTC(N)1↓ specificity as research-grade BspQI, manufactured to ISO 13485 and GMP standards and animal-origin free, with a US DMF on file (MF038306). Delivers high-efficiency, low-star-activity digestion — purpose-built for linearizing plasmid templates ahead of in vitro transcription in mRNA and biologics manufacturing.
Match the enzyme to the workflow
- Golden Gate / MoClo — BsaI + BbsI + Esp3I (BsmBI) for orthogonal, scarless one-pot assembly tiers.
- Routine subcloning — EcoRI, BamHI for standard sticky-end vector and insert preparation.
- Mutagenesis cleanup — DpnI to digest Dam-methylated parental template after site-directed mutagenesis.
- IVT / mRNA manufacturing — BspQI GMP-grade to linearize plasmid template before in vitro transcription.
Reproducible FuniCut™ restriction enzyme digestion protocol
A representative fast-digestion setup for the 37 °C FuniCut™ enzymes. All enzymes in the series use the universal 10× FuniCut™ Buffer (or the Color Buffer for direct gel loading), so the same recipe scales across single- and multi-enzyme digests. Volumes below are a typical starting point — adjust DNA input to your prep.
1 · Reaction setup (assemble on ice, in the order listed)
| Component | Plasmid DNA | PCR product | Genomic DNA |
|---|---|---|---|
| Nuclease-free ddH2O | 15 µL | 16 µL | 30 µL |
| 10× FuniCut™ Buffer (or Color Buffer) | 2 µL | 3 µL* | 5 µL |
| Substrate DNA | 2 µL (~1 µg) | 10 µL (~0.2 µg) | 10 µL (~5 µg) |
| FuniCut™ enzyme | 1 µL | 1 µL | 5 µL |
| Total volume | 20 µL | 30 µL | 50 µL |
*For purified PCR products. Unpurified products carry ionic strength — reduce 10× buffer to ~2 µL. Purify PCR products before digestion if the next step is cloning. Keep total enzyme volume ≤ 1/10 of the reaction to avoid glycerol-driven star activity.
2 · Run the digest
- Mix by gentle pipetting or swirling — do not vortex — then briefly centrifuge to collect droplets.
- Incubate at 37 °C: ~15 min (plasmid), 15–30 min (PCR product), or 30–60 min (genomic DNA).
- Optional: inactivate at 80 °C for 20 min (for heat-labile enzymes; BamHI is not heat-inactivatable — use column or phenol-chloroform cleanup).
- If the Color Buffer was used, load reaction products directly onto an agarose gel — no separate loading dye needed.
3 · Double & multiple digestion
Because the series shares one buffer, enzymes can be combined in a single tube. Use 1 µL of each enzyme and scale the reaction volume up as needed, keeping total enzyme ≤ 1/10 of the reaction. If the enzymes have different optimal temperatures, digest first with the lower-temperature enzyme, then add the higher-temperature enzyme and shift to its optimum.
Note on BspQI: the two BspQI options react at 50 °C for ~60 min (not 37 °C), so run them separately from the 37 °C FuniCut™ enzymes rather than in the same one-tube digest.
Storage & reaction notes
All enzymes ship cold and store at −25 °C to −15 °C — FuniCut™ enzymes for up to 2 years, BspQI (RUO) and BspQI GMP-grade for 1 year. Avoid repeated freeze-thaw. FuniCut™ enzymes are optimized for 37 °C; keep total enzyme volume to ≤1/10 of the reaction. For multi-enzyme digests with different optima, start at the lower temperature, then add the higher-temperature enzyme and shift up. BspQI enzymes react at 50 °C. Products are for research use only unless the GMP grade is specified.
Frequently asked questions
What are Type IIS restriction enzymes?
Type IIS restriction enzymes recognize an asymmetric DNA sequence and cut a fixed distance outside that sequence, rather than within it. This removes the recognition site from the digested product and lets fragments be joined scarlessly and directionally — the basis of Golden Gate and MoClo assembly. Examples include BsaI, BbsI, Esp3I (BsmBI), EarI, and BspQI.
How is a Type IIS enzyme different from a classic Type II enzyme?
A classic Type II enzyme (EcoRI, BamHI) cuts within a palindromic recognition site, so every fragment carries the same fixed overhang and the site remains in the product. A Type IIS enzyme cuts outside its site, so you can design custom, non-palindromic overhangs and produce a seamless junction.
Can I combine restriction enzymes in one buffer for a double digest?
Yes. Every FuniCut™ enzyme uses the same universal 10× FuniCut™ Buffer, so 37 °C enzymes can be combined in one tube for double or multiple digestion — 1 µL of each, keeping total enzyme ≤1/10 of the reaction. BspQI runs at 50 °C and should be run separately.
How fast are FuniCut™ fast restriction enzymes?
Complete digestion typically takes 5–15 minutes at 37 °C: about 15 min for plasmid DNA, 15–30 min for PCR products, and 30–60 min for genomic DNA — versus the classic 1–16 hour incubation.
Which enzyme should I use for Golden Gate / MoClo assembly?
BsaI is the workhorse for the first Golden Gate tier; BbsI and Esp3I (BsmBI) provide orthogonal recognition sites for hierarchical MoClo/Loop assembly. Using enzymes with different sites at each tier keeps assemblies scarless and directional.
Is there a GMP-grade restriction enzyme for mRNA / IVT manufacturing?
Yes. BspQI GMP-grade is manufactured to ISO 13485 and GMP standards, is animal-origin free, and has a US DMF on file (MF038306). It is purpose-built to linearize plasmid templates ahead of in vitro transcription in mRNA and biologics production.
Method references
- Engler C, Kandzia R, Marillonnet S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE. 2008;3(11):e3647. doi:10.1371/journal.pone.0003647 — the founding description of Golden Gate assembly using Type IIS enzymes (e.g., BsaI).
- Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE. 2011;6(2):e16765. doi:10.1371/journal.pone.0016765 — the MoClo standard, built on BsaI and BbsI/BpiI.
These references document the assembly methods that the Type IIS enzymes in this collection support; they are field context and do not constitute product-specific citations of the FuniCut™ line.
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