Sequence
Restriction sites
Find restriction sites on a linear or circular sequence, with a virtual digest, end-type filtering, type IIS support and a simulated gel.
Cutting enzymes
No restriction site found with these settings.
Simulated gel
Positions are one-based and point at the start of the recognition site. For type IIS enzymes the numbers in brackets are the cut distance from the end of the site. Methylation sensitivity is not modelled.
Formula
EcoRI: G^AATTC → a four-base 5′ overhang
- ^
- the cut position on the top strand
- number in brackets
- offset between the two strand cuts; positive means a 5′ overhang
- type IIS
- such as GGTCTC(1/5), which cuts outside its recognition site
Worked example
Picking enzymes for directional cloning into a vector
- insert and vector both need to be cut with two different enzymes
- Switch to unique cutters only, so enzymes that cut more than once drop out.
- Choose two enzymes with different overhangs so the insert can only go in one way round.
- Confirm that neither site occurs inside the insert itself.
Answer a pair of enzymes that each cut exactly once, inside the polylinker
Common mistakes
- Forgetting methylation sensitivity. Dam and Dcm methylation in E. coli blocks enzymes such as XbaI and ClaI; this tool does not model methylation.
- Leaving a plasmid marked as linear. Without the circular option, sites spanning the junction between the end and the start of the sequence are missed.
- Putting a restriction site at the very end of a primer with no extra bases. Most enzymes cannot cut a site flush with the end of a fragment; three to six extra bases are usually needed.
Frequently asked
Why do positions differ from another program?
Here positions are one-based and point at the start of the recognition site, not at the cut. Some programs report the cut position, a few bases further along.
What are type IIS enzymes for?
Because they cut outside their recognition site, they create any overhang you like and leave no scar in the product. That is the basis of Golden Gate and MoClo assembly.