Enzyme

Everything about one restriction enzyme against the sequence you have open: its recognition site, its cut offsets, every position it cuts, and all the names it goes by.

What it doesCopy Anchor Link

Clicking a cut site on the map, or a name in the cut-site filter, opens this. It is the detail view for one enzyme against the open sequence, and it answers the questions a map cannot:

• How many times does this cut? Stated in words as well as a number — single cutter, double cutter, triple cutter, n cutters, or does not cut — because that classification is the thing you are usually deciding on.
• Where exactly? One row per cut site, in order along the sequence, each giving the positions the two strands are cut at and whether the site was found on the forward strand, the reverse strand, or is palindromic — which most common six-base cutters are.
• What is the recognition sequence, and where does it cut relative to it? The site, plus the offsets at which the top and bottom strands are cut. Those two offsets are what determine the end you get: cut the top strand before the bottom one and the fragment carries a 5′ overhang, after it a 3′ overhang, at the same position a blunt end.
• What else is it called? Every alias, each marked with whether it is currently active in your enzyme groups — so an enzyme that appears to be missing is usually present under another name.

Selecting a group in the cut-site filter rather than a single enzyme opens the same panel for the group, listing its members and their sites.

When you'd use thisCopy Anchor Link

• Choosing a cloning site. Single cutter is what you are looking for, and the cut positions tell you what the ends will be.
• Working out whether two enzymes are compatible. Compatible ends need the same overhang in the same orientation; the cut offsets are what you compare, and the enzyme names tell you nothing.
• Designing a diagnostic digest, where the number of cuts and their spacing decide whether a gel will distinguish a correct clone from a wrong one.
• Checking a Type IIS site, where the enzyme cuts at a distance from the sequence it recognises, so the site you see on the map and the cut you will get are in different places.
• When an enzyme seems to be missing. Look at the alias list — the same enzyme is often stocked under a different supplier's name.

What this computesCopy Anchor Link

For one enzyme, or one group, against the open sequence:

• The cut count, and its classification in words — single cutter, double cutter, triple cutter, n cutters, does not cut — because that classification is usually the decision, not the number
• The recognition sequence and the top- and bottom-strand cut offsets, from REBASE. Those two offsets are what determine the end you get: top strand cut before the bottom leaves a 5′ overhang, after it a 3′ overhang, at the same position a blunt end — which is what you compare when asking whether two enzymes are compatible, since the names tell you nothing
• One row per cut site in order along the sequence, with both strands' cut positions and whether the site was found forward, reverse, or is palindromic — detected from the sequence rather than declared, by whether the forward and reverse recognition patterns are identical
• Type IIS cut positions reported separately from the site, because those enzymes cut at a fixed offset outside what they recognise, and the overhang they leave is whatever sequence sits at the cut — the property Golden Gate assembly is built on
• The alias list, each alias marked with whether it is active in your enzyme groups, so an enzyme that appears to be missing is usually present under another supplier's name

Topology changes the answer, correctly. On a circular sequence a site spanning the origin is a real site and is counted; a linear molecule has no such position, so the same bases can report a different count under the two topologies.

A hidden enzyme still reports its real count, not zero, so one can be looked up without first enabling its group.

What this is not: cut sites are sequence, not biochemistry. Star activity, methylation blocking (Dam, Dcm, CpG), buffer and temperature optima and heat inactivation are properties of the reaction, and an enzyme with a site here may still not cut your DNA in your tube.

Inputs and outputsCopy Anchor Link

In: one enzyme (or one group) and the open sequence, circular or linear.

Out: the cut count and its label, the recognition sequence, the top- and bottom-strand cut offsets, one row per cut site with its positions and strand, and the alias list with each alias's active state.

Cut sites are computed against the sequence in front of you, including for an enzyme that is hidden in your groups — a hidden enzyme reports its real count rather than zero, so you can look one up without first enabling it.

The scienceCopy Anchor Link

Recognition sequences and cut offsets come from REBASE, the restriction-enzyme database maintained by New England Biolabs, which is the authority for them. The same dataset drives the map, the cut-site filter, this panel, the digest tool and the cloning tools, so the sites you see, the cuts the tools make and the counts reported here agree with one another.

Palindromic is detected from the sequence, not declared. An enzyme whose forward and reverse recognition patterns are identical is palindromic and cuts the same way whichever strand it is read from; anything else has a strand, and it is reported.

Type IIS enzymes cut outside their recognition sequence, at a fixed offset from it, which is why the cut positions are reported separately from the site rather than implied by it — and why the overhang they leave is whatever sequence sits at the cut. That property is what Golden Gate assembly is built on.

On a circular sequence, sites that span the origin are real sites and are counted. A linear sequence has no such position, so the same molecule can report a different count depending on its topology, correctly.

LimitationsCopy Anchor Link

• Cut sites are sequence, not biochemistry. Star activity, methylation blocking (Dam, Dcm, CpG), buffer and temperature optima and heat inactivation are properties of the reaction. An enzyme with a site here may still not cut your DNA in your tube.
• What the map shows depends on your enzyme groups, even though this panel will describe a hidden enzyme correctly. Before concluding a sequence has no site for something, check which group is selected.
• A custom enzyme you defined is believed as entered. There is no authority to check it against, so its sites are as right as its definition.

RelatedCopy Anchor Link

In this group: Edit annotation · Insert sequence · Merge features · Manage enzymes · Dot plot · Dot plot grid · Melt curve · Amplification curves · Population genetics · RNA-seq · Site avoidance heat map · Protein coverage · Reference-gene stability · Digital PCR · Codon usage matrix · Pairwise distance distribution · Nearest-neighbor distances · GC content distribution · Base composition per cycle · Pairwise distance grid · Per-cycle channel occupancy · Grammar assembly plan · Gibson assembly plan · Assembly plan build order · Golden Gate assembly plan · Guide track · Primer binding track · Hydropathy profile · Charge vs pH · Amino-acid composition · Hydrophobic moment · Optimization profiles · Digest gel · Virtual gel · Initiation-window fold · Antibody domain map · Electropherogram · Circuit netlist · Circuit transfer functions · Circuit construct · Construction graph · Protocol · Read Quality Profile · Read Alignment · Raw Signal · Verify vs Reference · Chromosomal microarray · Variant calls · Small molecule