Predict restriction enzyme cut sites, fragment sizes, sticky ends, blunt ends, and compatible overhangs from a DNA sequence. Use the tool for plasmid maps, PCR insert screening, cloning lessons, and quick checks before a wet-lab digest. It updates live as you change the sequence, DNA topology, or enzyme set.
Paste a DNA sequence, choose linear or circular topology, and select enzymes to see cut coordinates and predicted bands immediately.
Start with a screening digest, a plasmid multiple cloning site, or a compatible-end example.
Paste a plasmid, PCR product, insert, or synthetic DNA sequence. Spaces and numbers are ignored.
DNA topology
Length
86
bp
Selected
2
enzymes
Cuts
2
positions
Fragments
3
bands
Select one enzyme for a diagnostic digest or multiple enzymes for a double-digest style map.
Live digest result
The predicted digest creates 3 fragments from a linear 86 bp molecule.
Unique
2
No cut
0
Groups
0
Digest notes
Cut marks are placed by base-pair coordinate on the sequence or circular plasmid map.
Larger fragments appear closer to the top of a simple agarose-gel style view.
| Enzyme | Site | Cut | End |
|---|---|---|---|
| EcoRI | 6–11 | 6 | 5′ overhang |
| HindIII | 71–76 | 71 | 5′ overhang |
A restriction enzyme digest predictor turns a DNA sequence into a virtual restriction map. It scans for recognition sequences, places cut sites at enzyme-specific coordinates, and calculates the DNA fragments that would appear after digestion. The result answers a practical question: which enzymes cut this sequence, and what band sizes should I expect?
Restriction endonucleases recognize defined DNA motifs and cut DNA in predictable positions. Some enzymes leave cohesive overhangs, while others create blunt ends. NEB’s compatible cohesive-end chart shows why enzymes such as BamHI and BglII can create ligation-compatible ends even though they recognize different DNA sequences. Review the NEB compatible ends chart.
In molecular cloning, the same logic helps screen plasmids, release inserts, check vector maps, and plan directional ligation. If you amplify an insert first, confirm the amplicon length with the PCR Product Size Calculator before you interpret restriction bands.
Enter the insert, plasmid, PCR product, or synthetic sequence using A, C, G, and T letters.
Select linear DNA for PCR products and inserts, or circular plasmid for closed vector maps.
Tick one or more enzymes such as EcoRI, BamHI, HindIII, NotI, SmaI, or PstI.
Use the map, cut-site table, and fragment bars to interpret the predicted digest pattern.
Enter the DNA strand in 5′ to 3′ order when possible. The current enzyme list uses exact recognition sequences, so ambiguous bases such as N, R, and Y are removed before matching.
| Tool component | Purpose | How to interpret it |
|---|---|---|
| DNA sequence box | Accepts plasmid, insert, PCR product, or synthetic DNA sequence. | Longer sequences can create more sites, especially for four-base cutters. |
| Linear/circular selector | Controls whether the molecule has ends or forms a closed plasmid. | One cut linearizes a circular plasmid but splits a linear insert into two pieces. |
| Enzyme panel | Lists recognition motifs, cut types, overhangs, and selected enzymes. | Unique cutters help mapping; compatible overhang groups help ligation planning. |
| Restriction map preview | Places enzyme labels on the DNA molecule by base-pair coordinate. | Clusters of marks can predict small fragments or complex gel patterns. |
| Fragment-size bars | Ranks predicted digest fragments from largest to smallest. | Bands below 50 bp may run off many agarose gels or stain weakly. |
Most classroom digest problems focus on Type II restriction enzymes because they cut at predictable positions near or inside the recognition sequence. The table below shows common enzymes included in the calculator. For primer and insert checks, combine this digest map with an oligo sequence analysis workflow.
| Enzyme | Recognition sequence | Predicted end | Typical use |
|---|---|---|---|
| EcoRI | GAATTC | 5′ AATT overhang | Introductory cloning and insert release maps |
| BamHI | GGATCC | 5′ GATC overhang | Directional cloning and compatible-end examples |
| HindIII | AAGCTT | 5′ AGCT overhang | Vector screening and multiple cloning sites |
| PstI | CTGCAG | 3′ TGCA overhang | Distinguishing 3′ overhang behavior from 5′ cutters |
| SmaI | CCCGGG | Blunt | Blunt-end ligation examples |
A 900 bp linear PCR insert contains one EcoRI site at cut position 80 and one HindIII site at cut position 840. The digest creates fragments of 80 bp, 760 bp, and 60 bp. The 760 bp band carries most of the insert and should dominate the gel image.
This result can confirm that restriction sites were added to primer tails. Use the GC Content Calculator when you need a quick sequence-composition check before comparing primers or synthetic inserts.
A 4,200 bp circular plasmid has a BamHI cut at 620 bp and an XhoI cut at 2,900 bp. The double digest predicts two fragments: 2,280 bp and 1,920 bp. A gel with two bands near those sizes supports the expected plasmid map.
If the plasmid contains only one selected site, the digest produces one linear band equal to the plasmid size. That pattern confirms linearization but does not release a separate insert fragment.
Fragment sizes answer different questions depending on the experimental design. One band from a circular plasmid usually means one selected cut site. Two bands can show that the enzyme pair cuts on both sides of an insert. More bands may indicate repeated sites, unexpected rearrangement, or an enzyme that cuts inside the insert.
End type answers a different question. EcoRI and BamHI create 5′ overhangs, PstI and KpnI create 3′ overhangs, and SmaI produces blunt termini. Compatible-end predictions help you see which enzyme pairs can ligate, but they do not prove that the ligated junction will remain digestible.
This calculator predicts sequence-based cleavage. It does not model methylation sensitivity, star activity, buffer compatibility, enzyme unit concentration, incubation time, DNA supercoiling, or incomplete digestion. Supplier data sheets still matter for a real bench protocol.
Very small fragments can escape detection on routine agarose gels. Similar fragment sizes can also merge into one visible band. Use predicted fragment sizes as a planning guide, then compare them with a DNA ladder and lab conditions.
Use these tools with restriction digest planning when you design primers, verify products, or check sequence composition.