Input - up to 200,000 bp, from local sequence
file, GenBank or by cutting and pasting.
Enzymes - default searches with NEB enzymes
(and all neoschizomers) - options include all commercial or all known specificities.
ORF display - default is >100 amino acids,
Met to stop codon, non-overlapping.
Navigating - mouse over the main features
for additional information, click for yet more options.
Zooming - one mark and zoom gives 10-fold
increase, two marks define a region.
Privacy - sequences submitted to NEBcutter
are maintained locally for 2 days and then discarded. No-one at NEB
checks them for content.
The program accepts an input sequence, which can be pasted
in, picked up from a local file or retrieved from NCBI as a Genbank file
via its accession number. It calculates all long open reading frames and
then displays the sequence, the long ORFs and all NEB enzymes that cut
it just once. It also shows the enzymes which could be used in a complete
digest to excise each open reading frame that it finds. From the initial
display there are options to go further, including custom digestion with
enzymes of your choice and various displays of the digest. One feature
that is implemented on some pages is that moving the mouse over an enzyme
name will produce a box with the recognition sequence and will also underline
the bases of that recognition within the display. On the digest, moving
the mouse on to the bands will display the fragment length and the enzyme
that produced each end of the fragment.
Ambiguous nucleotides are allowed in the input sequence, up to
5 in any 20 nt window. If there are more then a warning message is displayed
and all degenerate bases are ignored in the sequence.
Recognition sites which depend on the precise sequence but overlap a
degenerate base are marked by putting the enzyme name in parentheses.
When the program searches for ORFs, by default it uses the
bacterial genetic code (which can be changed on the opening page) to identify
start and stop codons. If two ORFs significantly overlap then the smaller
one is dropped, keeping only the longest, non-overlapping ones. Since not
only ATG is treated as an initiation codon, some ORFs may be predicted to be
longer than they actually are. In this case you can either
choose the "Standard" genetic code, which has less alternative start codons,
or use the "edit ORF" feature to specify the correct coordinates.
NEBcutter incorporates everything that is known about the methylation
sensitivity of any of the enzymes displayed when they overlap say a Dam
site or a Dcm site as well as CpG, EcoK and EcoB sites. When theoretical
digests are performed the results include a comparison of the effects of
methylation, highlighting the bands that shift.
We have tried to make most of the options intuitive with buttons
to provide obvious analyses. Please feel free to give us feedback by using
the "comment" feature in the top right hand
corner of each page.
Vincze and Richard