Find restriction enzymes producing a specific overhang


restover identifies restriction enzymes from the REBASE database that create the specified overhang sequence when they cut the input nucleotide sequence(s). It writes an output file which shows the base number, restriction enzyme name, recognition site and cut positions. There are several options to control exactly what sites are reported and the format of the output file. Optionally, output in HTML may be generated.


Here is a sample session with restover

% restover 
Find restriction enzymes producing a specific overhang
Input nucleotide sequence(s): tembl:x65923
Overlap sequence: cg
Output file [x65923.restover]: 

Go to the input files for this example
Go to the output files for this example

Command line arguments

   Standard (Mandatory) qualifiers:
  [-sequence]          seqall     Nucleotide sequence(s) filename and optional
                                  format, or reference (input USA)
  [-seqcomp]           string     Overlap sequence (Any string is accepted)
  [-outfile]           outfile    [*.restover] Output file name

   Additional (Optional) qualifiers: (none)
   Advanced (Unprompted) qualifiers:
   -datafile           datafile   Restriction enzyme data file (optional)
   -min                integer    [1] Minimum cuts per RE (Integer from 1 to
   -max                integer    [2000000000] Maximum cuts per RE (Integer up
                                  to 2000000000)
   -single             boolean    [N] Force single site only cuts
   -threeprime         boolean    [N] Use 3' overhang e.g. BamHI has CTAG as a
                                  5' overhang, and ApaI has CCGG as 3'
   -[no]blunt          boolean    [Y] Allow blunt end cutters
   -[no]sticky         boolean    [Y] Allow sticky end cutters
   -[no]ambiguity      boolean    [Y] Allow ambiguous matches
   -plasmid            boolean    [N] Allow circular DNA
   -[no]commercial     boolean    [Y] Only enzymes with suppliers
   -html               boolean    [N] Create HTML output
   -[no]limit          boolean    [Y] Limits reports to one isoschizomer
   -alphabetic         boolean    [N] Sort output alphabetically
   -fragments          boolean    [N] Show fragment lengths

   Associated qualifiers:

   "-sequence" associated qualifiers
   -sbegin1            integer    Start of each sequence to be used
   -send1              integer    End of each sequence to be used
   -sreverse1          boolean    Reverse (if DNA)
   -sask1              boolean    Ask for begin/end/reverse
   -snucleotide1       boolean    Sequence is nucleotide
   -sprotein1          boolean    Sequence is protein
   -slower1            boolean    Make lower case
   -supper1            boolean    Make upper case
   -sformat1           string     Input sequence format
   -sdbname1           string     Database name
   -sid1               string     Entryname
   -ufo1               string     UFO features
   -fformat1           string     Features format
   -fopenfile1         string     Features file name

   "-outfile" associated qualifiers
   -odirectory3        string     Output directory

   General qualifiers:
   -auto               boolean    Turn off prompts
   -stdout             boolean    Write first file to standard output
   -filter             boolean    Read first file from standard input, write
                                  first file to standard output
   -options            boolean    Prompt for standard and additional values
   -debug              boolean    Write debug output to program.dbg
   -verbose            boolean    Report some/full command line options
   -help               boolean    Report command line options. More
                                  information on associated and general
                                  qualifiers can be found with -help -verbose
   -warning            boolean    Report warnings
   -error              boolean    Report errors
   -fatal              boolean    Report fatal errors
   -die                boolean    Report dying program messages

Standard (Mandatory) qualifiers Allowed values Default
(Parameter 1)
Nucleotide sequence(s) filename and optional format, or reference (input USA) Readable sequence(s) Required
(Parameter 2)
Overlap sequence Any string is accepted An empty string is accepted
(Parameter 3)
Output file name Output file <*>.restover
Additional (Optional) qualifiers Allowed values Default
Advanced (Unprompted) qualifiers Allowed values Default
-datafile Restriction enzyme data file (optional) Data file File in the data file path
-min Minimum cuts per RE Integer from 1 to 1000 1
-max Maximum cuts per RE Integer up to 2000000000 2000000000
-single Force single site only cuts Boolean value Yes/No No
-threeprime Use 3' overhang e.g. BamHI has CTAG as a 5' overhang, and ApaI has CCGG as 3' overhang. Boolean value Yes/No No
-[no]blunt Allow blunt end cutters Boolean value Yes/No Yes
-[no]sticky Allow sticky end cutters Boolean value Yes/No Yes
-[no]ambiguity Allow ambiguous matches Boolean value Yes/No Yes
-plasmid Allow circular DNA Boolean value Yes/No No
-[no]commercial Only enzymes with suppliers Boolean value Yes/No Yes
-html Create HTML output Boolean value Yes/No No
-[no]limit Limits reports to one isoschizomer Boolean value Yes/No Yes
-alphabetic Sort output alphabetically Boolean value Yes/No No
-fragments Show fragment lengths Boolean value Yes/No No

Input file format

restover reads in a normal nucleic acid sequence USA.

Input files for usage example

'tembl:x65923' is a sequence entry in the example nucleic acid database 'tembl'

Database entry: tembl:x65923

ID   X65923; SV 1; linear; mRNA; STD; HUM; 518 BP.
AC   X65923;
DT   13-MAY-1992 (Rel. 31, Created)
DT   18-APR-2005 (Rel. 83, Last updated, Version 11)
DE   H.sapiens fau mRNA
KW   fau gene.
OS   Homo sapiens (human)
OC   Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia;
OC   Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae;
OC   Homo.
RN   [1]
RP   1-518
RA   Michiels L.M.R.;
RT   ;
RL   Submitted (29-APR-1992) to the EMBL/GenBank/DDBJ databases.
RL   L.M.R. Michiels, University of Antwerp, Dept of Biochemistry,
RL   Universiteisplein 1, 2610 Wilrijk, BELGIUM
RN   [2]
RP   1-518
RX   PUBMED; 8395683.
RA   Michiels L., Van der Rauwelaert E., Van Hasselt F., Kas K., Merregaert J.;
RT   " fau cDNA encodes a ubiquitin-like-S30 fusion protein and is expressed as
RT   an antisense sequences in the Finkel-Biskis-Reilly murine sarcoma virus";
RL   Oncogene 8(9):2537-2546(1993).
DR   H-InvDB; HIT000322806.
FH   Key             Location/Qualifiers
FT   source          1..518
FT                   /organism="Homo sapiens"
FT                   /chromosome="11q"
FT                   /map="13"
FT                   /mol_type="mRNA"
FT                   /clone_lib="cDNA"
FT                   /clone="pUIA 631"
FT                   /tissue_type="placenta"
FT                   /db_xref="taxon:9606"
FT   misc_feature    57..278
FT                   /note="ubiquitin like part"
FT   CDS             57..458
FT                   /gene="fau"
FT                   /db_xref="GDB:135476"
FT                   /db_xref="GOA:P35544"
FT                   /db_xref="GOA:P62861"
FT                   /db_xref="HGNC:3597"
FT                   /db_xref="UniProtKB/Swiss-Prot:P35544"
FT                   /db_xref="UniProtKB/Swiss-Prot:P62861"
FT                   /protein_id="CAA46716.1"
FT   misc_feature    98..102
FT                   /note="nucleolar localization signal"
FT   misc_feature    279..458
FT                   /note="S30 part"
FT   polyA_signal    484..489
FT   polyA_site      509
SQ   Sequence 518 BP; 125 A; 139 C; 148 G; 106 T; 0 other;
     ttcctctttc tcgactccat cttcgcggta gctgggaccg ccgttcagtc gccaatatgc        60
     agctctttgt ccgcgcccag gagctacaca ccttcgaggt gaccggccag gaaacggtcg       120
     cccagatcaa ggctcatgta gcctcactgg agggcattgc cccggaagat caagtcgtgc       180
     tcctggcagg cgcgcccctg gaggatgagg ccactctggg ccagtgcggg gtggaggccc       240
     tgactaccct ggaagtagca ggccgcatgc ttggaggtaa agttcatggt tccctggccc       300
     gtgctggaaa agtgagaggt cagactccta aggtggccaa acaggagaag aagaagaaga       360
     agacaggtcg ggctaagcgg cggatgcagt acaaccggcg ctttgtcaac gttgtgccca       420
     cctttggcaa gaagaagggc cccaatgcca actcttaagt cttttgtaat tctggctttc       480
     tctaataaaa aagccactta gttcagtcaa aaaaaaaa                               518

Output file format

Output files for usage example

File: x65923.restover

# Restrict of X65923 from 1 to 518
# Minimum cuts per enzyme: 1
# Maximum cuts per enzyme: 2000000000
# Minimum length of recognition site: 2
# Number of hits with any overlap: 54
# Base Number	Enzyme		Site		5'	3'	[5'	3']
	11	TaqI            TCGA            11	13	
	28	AciI            CCGC            25	27	
	38	AciI            CCGC            38	40	
	44	BceAI           ACGGC           25	27	
	71	AciI            CCGC            71	73	
	73	Hin6I           GCGC            73	75	
	94	TaqI            TCGA            94	96	
	103	HpaII           CCGG            103	105	
	162	HpaII           CCGG            162	164	
	190	Hin6I           GCGC            190	192	
	192	Hin6I           GCGC            192	194	
	225	BsrI            ACTGG           221	219	
	229	AciI            CCGC            226	228	
	263	AciI            CCGC            263	265	
	380	AciI            CCGC            377	379	
	383	AciI            CCGC            380	382	
	395	HpaII           CCGG            395	397	
	398	Hin6I           GCGC            398	400	
	408	AclI            AACGTT          409	411	
	409	MaeII           ACGT            409	411	

The output from restover is a simple text one. The base number, restriction enzyme name, recognition site and cut positions are shown. Note that cuts are always to the right of the residue shown and that 5' cuts are referred to by their associated 3' number sequence. The program reports enzymes that cut at two or four sites.

Data files

EMBOSS data files are distributed with the application and stored in the standard EMBOSS data directory, which is defined by the EMBOSS environment variable EMBOSS_DATA.

To see the available EMBOSS data files, run:

% embossdata -showall

To fetch one of the data files (for example 'Exxx.dat') into your current directory for you to inspect or modify, run:

% embossdata -fetch -file Exxx.dat

Users can provide their own data files in their own directories. Project specific files can be put in the current directory, or for tidier directory listings in a subdirectory called ".embossdata". Files for all EMBOSS runs can be put in the user's home directory, or again in a subdirectory called ".embossdata".

The directories are searched in the following order:

The EMBOSS REBASE restriction enzyme data files are stored in directory 'data/REBASE/*' under the EMBOSS installation directory.

These files must first be set up using the program 'rebaseextract'. Running 'rebaseextract' may be the job of your system manager.

The data files are stored in the REBASE directory of the standard EMBOSS data directory. The names are:

The column information is described at the top of the data files

The reported enzyme from any one group of isoschizomers (the prototype) is specified in the REBASE database and the information is held in the data file 'embossre.equ'. You may edit this file to set your own preferred prototype, if you wish.

The format of the file "embossre.equ" is
Enzyme-name Prototype-name

i.e. two columns of enzyme names separated by a space. The first name of the pair of enzymes is the name that is not preferred and the second is the preferred (prototype) name.


The Restriction Enzyme database (REBASE) is a collection of information about restriction enzymes and related proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. DNA methyltransferases, homing endonucleases, nicking enzymes, specificity subunits and control proteins are also included. Most recently, putative DNA methyltransferases and restriction enzymes, as predicted from analysis of genomic sequences, are also listed.

The home page of REBASE is:

Several criteria may be set to control what sites are reported: -min, -max, -single (minimum or maximum number of cuts, or single site cuts only. -blunt (enzymes which cut at the same position on the forward and reverse strands). -sticky (enzymes which cut at different positions on the forward and reverse strands, leaving an overhang). -ambiguity (enzymes which have one or more N ambiguity codes in their pattern). -commercial (enzymes with a commercial supplier). -plasmid (allows searches for restriction enzyme recognition site and cut postions that span the end of the sequence).

By default, only one enzyme of any group of isoschizomers (enzymes that have the same recognition site and cut positions) is reported. This behaviour can be changed by specifying '-nolimit', in which case all isoschizomers are reported. The default behaviour uses the representative enzyme of an isoschizomer group (the prototype) which is specified in the EMBOSS data file embossre.equ. This file is generated from the REBASE database by running rebaseextract. You may edit this file to set your own preferred prototype,if you wish.




restover uses the EMBOSS REBASE restriction enzyme data files stored in directory data/REBASE/* under the EMBOSS installation directory. These files must first be set up using the program rebaseextract. Running rebaseextract may be the job of your system manager.

Diagnostic Error Messages


Exit status

It always exits with status 0.

Known bugs


See also

Program name Description
recoder Find restriction sites to remove (mutate) with no translation change
redata Retrieve information from REBASE restriction enzyme database
remap Display restriction enzyme binding sites in a nucleotide sequence
restrict Report restriction enzyme cleavage sites in a nucleotide sequence
showseq Displays sequences with features in pretty format
silent Find restriction sites to insert (mutate) with no translation change


Bernd Jagla (bernd ©
Cellular Biochemistry and Biophysics Program, Rockefeller Research Laboratories, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 251,New York, NY 10021.


Written (Jan 2001) - Bernd Jagla

Target users

This program is intended to be used by everyone and everything, from naive users to embedded scripts.