#
# BioPerl module for Bio::Variation::VariantI
#
# Please direct questions and support issues to <bioperl-l@bioperl.org>
#
# Cared for by Heikki Lehvaslaiho <heikki-at-bioperl-dot-org>
#
# Copyright Heikki Lehvaslaiho
#
# You may distribute this module under the same terms as perl itself
# POD documentation - main docs before the code
=head1 NAME
Bio::Variation::VariantI - Sequence Change SeqFeature abstract class
=head1 SYNOPSIS
#get Bio::Variant::VariantI somehow
print $var->restriction_changes, "\n";
foreach $allele ($var->each_Allele) {
#work on Bio::Variation::Allele objects
}
=head1 DESCRIPTION
This superclass defines common methods to basic sequence changes. The
instantiable classes Bio::Variation::DNAMutation,
Bio::Variation::RNAChange and Bio::Variation::AAChange use them.
See L<Bio::Variation::DNAMutation>, L<Bio::Variation::RNAChange>,
and L<Bio::Variation::AAChange> for more information.
These classes store information, heavy computation to detemine allele
sequences is done elsewhere.
The database cross-references are implemented as
Bio::Annotation::DBLink objects. The methods to access them are
defined in Bio::DBLinkContainerI. See L<Bio::Annotation::DBLink>
and L<Bio::DBLinkContainerI> for details.
Bio::Variation::VariantI redifines and extends
Bio::SeqFeature::Generic for sequence variations. This class
describes specific sequence change events. These events are always
from a specific reference sequence to something different. See
L<Bio::SeqFeature::Generic> for more information.
IMPORTANT: The notion of reference sequence permeates all
Bio::Variation classes. This is especially important to remember when
dealing with Alleles. In a polymorphic site, there can be a large
number of alleles. One of then has to be selected to be the reference
allele (allele_ori). ALL the rest has to be passed to the Variant
using the method add_Allele, including the mutated allele in a
canonical mutation. The IO modules and generated attributes depend on
it. They ignore the allele linked to using allele_mut and circulate
each Allele returned by each_Allele into allele_mut and calculate
the changes between that and allele_ori.
=head1 FEEDBACK
=head2 Mailing Lists
User feedback is an integral part of the evolution of this and other
Bioperl modules. Send your comments and suggestions preferably to the
Bioperl mailing lists Your participation is much appreciated.
bioperl-l@bioperl.org - General discussion
http://bioperl.org/wiki/Mailing_lists - About the mailing lists
=head2 Support
Please direct usage questions or support issues to the mailing list:
I<bioperl-l@bioperl.org>
rather than to the module maintainer directly. Many experienced and
reponsive experts will be able look at the problem and quickly
address it. Please include a thorough description of the problem
with code and data examples if at all possible.
=head2 Reporting Bugs
Report bugs to the Bioperl bug tracking system to help us keep track
the bugs and their resolution. Bug reports can be submitted via the
web:
https://redmine.open-bio.org/projects/bioperl/
=head1 AUTHOR - Heikki Lehvaslaiho
Email: heikki-at-bioperl-dot-org
=head1 APPENDIX
The rest of the documentation details each of the object
methods. Internal methods are usually preceded with a _
=cut
# Let the code begin...
package Bio::Variation::VariantI;
use strict;
# Object preamble - inheritance
use base qw(Bio::Root::Root Bio::SeqFeature::Generic Bio::DBLinkContainerI);
=head2 id
Title : id
Usage : $obj->id
Function:
Read only method. Returns the id of the variation object.
The id is the id of the first DBLink object attached to this object.
Example :
Returns : scalar
Args : none
=cut
sub id {
my ($self) = @_;
my @ids = $self->each_DBLink;
my $id = $ids[0] if scalar @ids > 0;
return $id->database. "::". $id->primary_id if $id;
}
=head2 add_Allele
Title : add_Allele
Usage : $self->add_Allele($allele)
Function:
Adds one Bio::Variation::Allele into the list of alleles.
Note that the method forces the convention that nucleotide
sequence is in lower case and amino acds are in upper
case.
Example :
Returns : 1 when succeeds, 0 for failure.
Args : Allele object
=cut
sub add_Allele {
my ($self,$value) = @_;
if (defined $value) {
if( ! $value->isa('Bio::Variation::Allele') ) {
my $com = ref $value;
$self->throw("Is not a Allele object but a [$com]");
return 0;
} else {
if ( $self->isa('Bio::Variation::AAChange') ) {
$value->seq( uc $value->seq) if $value->seq;
} else {
$value->seq( lc $value->seq) if $value->seq;
}
push(@{$self->{'alleles'}},$value);
$self->allele_mut($value); #????
return 1;
}
} else {
return 0;
}
}
=head2 each_Allele
Title : alleles
Usage : $obj->each_Allele();
Function:
Returns a list of Bio::Variation::Allele objects
Example :
Returns : list of Alleles
Args : none
=cut
sub each_Allele{
my ($self,@args) = @_;
return @{$self->{'alleles'}};
}
=head2 isMutation
Title : isMutation
Usage : print join('/', $obj->each_Allele) if not $obj->isMutation;
Function:
Returns or sets the boolean value indicating that the
variant descibed is a canonical mutation with two alleles
assinged to be the original (wild type) allele and mutated
allele, respectively. If this value is not set, it is
assumed that the Variant descibes polymorphisms.
Returns : a boolean
=cut
sub isMutation {
my ($self,$value) = @_;
if (defined $value) {
if ($value ) {
$self->{'isMutation'} = 1;
} else {
$self->{'isMutation'} = 0;
}
}
return $self->{'isMutation'};
}
=head2 allele_ori
Title : allele_ori
Usage : $obj->allele_ori();
Function:
Links to and returns the Bio::Variation::Allele object.
If value is not set, returns false. All other Alleles are
compared to this.
Amino acid sequences are stored in upper case characters,
others in lower case.
Example :
Returns : string
Args : string
See L<Bio::Variation::Allele> for more.
=cut
sub allele_ori {
my ($self,$value) = @_;
if( defined $value) {
if ( ! ref $value || ! $value->isa('Bio::Variation::Allele')) {
$self->throw("Value is not Bio::Variation::Allele but [$value]");
} else {
if ( $self->isa('Bio::Variation::AAChange') ) {
$value->seq( uc $value->seq) if $value->seq;
} else {
$value->seq( lc $value->seq) if $value->seq;
}
$self->{'allele_ori'} = $value;
}
}
return $self->{'allele_ori'};
}
=head2 allele_mut
Title : allele_mut
Usage : $obj->allele_mut();
Function:
Links to and returns the Bio::Variation::Allele
object. Sets and returns the mutated allele sequence.
If value is not set, returns false.
Amino acid sequences are stored in upper case characters,
others in lower case.
Example :
Returns : string
Args : string
See L<Bio::Variation::Allele> for more.
=cut
sub allele_mut {
my ($self,$value) = @_;
if( defined $value) {
if ( ! ref $value || ! $value->isa('Bio::Variation::Allele')) {
$self->throw("Value is not Bio::Variation::Allele but [$value]");
} else {
if ( $self->isa('Bio::Variation::AAChange') ) {
$value->seq( uc $value->seq) if $value->seq;
} else {
$value->seq( lc $value->seq) if $value->seq;
}
$self->{'allele_mut'} = $value;
}
}
return $self->{'allele_mut'};
}
=head2 length
Title : length
Usage : $obj->length();
Function:
Sets and returns the length of the affected original
allele sequence. If value is not set, returns false == 0.
Value 0 means that the variant position is before the
start=end sequence position. (Value 1 would denote a point
mutation). This follows the convension to report an
insertion (2insT) in equivalent way to a corresponding
deletion (2delT) (Think about indel polymorpism ATC <=> AC
where the origianal state is not known ).
Example :
Returns : string
Args : string
=cut
sub length {
my ($self,$value) = @_;
if ( defined $value) {
$self->{'length'} = $value;
}
if ( ! exists $self->{'length'} ) {
return 0;
}
return $self->{'length'};
}
=head2 upStreamSeq
Title : upStreamSeq
Usage : $obj->upStreamSeq();
Function:
Sets and returns upstream flanking sequence string. If
value is not set, returns false. The sequence should be
>=25 characters long, if possible.
Example :
Returns : string or false
Args : string
=cut
sub upStreamSeq {
my ($self,$value) = @_;
if( defined $value) {
$self->{'upstreamseq'} = $value;
}
return $self->{'upstreamseq'};
}
=head2 dnStreamSeq
Title : dnStreamSeq
Usage : $obj->dnStreamSeq();
Function:
Sets and returns dnstream flanking sequence string. If
value is not set, returns false. The sequence should be
>=25 characters long, if possible.
Example :
Returns : string or false
Args : string
=cut
sub dnStreamSeq {
my ($self,$value) = @_;
if( defined $value) {
$self->{'dnstreamseq'} = $value;
}
return $self->{'dnstreamseq'};
}
=head2 label
Title : label
Usage : $obj->label();
Function:
Sets and returns mutation event label(s). If value is not
set, or no argument is given returns false. Each
instantiable class needs to implement this method. Valid
values are listed in 'Mutation event controlled vocabulary' in
http://www.ebi.ac.uk/mutations/recommendations/mutevent.html.
Example :
Returns : string
Args : string
=cut
sub label {
my ($self,$value) = @_;
$self->throw_not_implemented();
}
=head2 status
Title : status
Usage : $obj->status()
Function:
Returns the status of the sequence change object.
Valid values are: 'suspected' and 'proven'
Example : $obj->status('proven');
Returns : scalar
Args : valid string (optional, for setting)
=cut
sub status {
my ($self,$value) = @_;
my %status = (suspected => 1,
proven => 1
);
if( defined $value) {
$value = lc $value;
if ($status{$value}) {
$self->{'status'} = $value;
}
else {
$self->throw("$value is not valid status value!");
}
}
if( ! exists $self->{'status'} ) {
return "$self";
}
return $self->{'status'};
}
=head2 proof
Title : proof
Usage : $obj->proof()
Function:
Returns the proof of the sequence change object.
Valid values are: 'computed' and 'experimental'.
Example : $obj->proof('computed');
Returns : scalar
Args : valid string (optional, for setting)
=cut
sub proof {
my ($self,$value) = @_;
my %proof = (computed => 1,
experimental => 1
);
if( defined $value) {
$value = lc $value;
if ($proof{$value}) {
$self->{'proof'} = $value;
} else {
$self->throw("$value is not valid proof value!");
}
}
return $self->{'proof'};
}
=head2 region
Title : region
Usage : $obj->region();
Function:
Sets and returns the name of the sequence region type or
protein domain at this location. If value is not set,
returns false.
Example :
Returns : string
Args : string
=cut
sub region {
my ($self,$value) = @_;
if( defined $value) {
$self->{'region'} = $value;
}
return $self->{'region'};
}
=head2 region_value
Title : region_value
Usage : $obj->region_value();
Function:
Sets and returns the name of the sequence region_value or
protein domain at this location. If value is not set,
returns false.
Example :
Returns : string
Args : string
=cut
sub region_value {
my ($self,$value) = @_;
if( defined $value) {
$self->{'region_value'} = $value;
}
return $self->{'region_value'};
}
=head2 region_dist
Title : region_dist
Usage : $obj->region_dist();
Function:
Sets and returns the distance tot the closest region
(i.e. intro/exon or domain) boundary. If distance is not
set, returns false.
Example :
Returns : integer
Args : integer
=cut
sub region_dist {
my ($self,$value) = @_;
if( defined $value) {
if ( not $value =~ /^[+-]?\d+$/ ) {
$self->throw("[$value] for region_dist has to be an integer\n");
} else {
$self->{'region_dist'} = $value;
}
}
return $self->{'region_dist'};
}
=head2 numbering
Title : numbering
Usage : $obj->numbering()
Function:
Returns the numbering chema used locating sequnce features.
Valid values are: 'entry' and 'coding'
Example : $obj->numbering('coding');
Returns : scalar
Args : valid string (optional, for setting)
=cut
sub numbering {
my ($self,$value) = @_;
my %numbering = (entry => 1,
coding => 1
);
if( defined $value) {
$value = lc $value;
if ($numbering{$value}) {
$self->{'numbering'} = $value;
}
else {
$self->throw("'$value' is not a valid for numbering!");
}
}
if( ! exists $self->{'numbering'} ) {
return "$self";
}
return $self->{'numbering'};
}
=head2 mut_number
Title : mut_number
Usage : $num = $obj->mut_number;
: $num = $obj->mut_number($number);
Function:
Returns or sets the number identifying the order in which the
mutation has been issued. Numbers shouldstart from 1.
If the number has never been set, the method will return ''
If you want the output from IO modules look nice and, for
multivariant/allele variations, make sense you better set
this attribute.
Returns : an integer
=cut
sub mut_number {
my ($self,$value) = @_;
if (defined $value) {
$self->{'mut_number'} = $value;
}
unless (exists $self->{'mut_number'}) {
return ('');
} else {
return $self->{'mut_number'};
}
}
=head2 SeqDiff
Title : SeqDiff
Usage : $mutobj = $obj->SeqDiff;
: $mutobj = $obj->SeqDiff($objref);
Function:
Returns or sets the link-reference to the umbrella
Bio::Variation::SeqDiff object. If there is no link,
it will return undef
Note: Adding a variant into a SeqDiff object will
automatically set this value.
Returns : an obj_ref or undef
See L<Bio::Variation::SeqDiff> for more information.
=cut
sub SeqDiff {
my ($self,$value) = @_;
if (defined $value) {
if( ! $value->isa('Bio::Variation::SeqDiff') ) {
$self->throw("Is not a Bio::Variation::SeqDiff object but a [$value]");
return;
}
else {
$self->{'seqDiff'} = $value;
}
}
unless (exists $self->{'seqDiff'}) {
return;
} else {
return $self->{'seqDiff'};
}
}
=head2 add_DBLink
Title : add_DBLink
Usage : $self->add_DBLink($ref)
Function: adds a link object
Example :
Returns :
Args :
=cut
sub add_DBLink{
my ($self,$com) = @_;
if( $com && ! $com->isa('Bio::Annotation::DBLink') ) {
$self->throw("Is not a link object but a [$com]");
}
$com && push(@{$self->{'link'}},$com);
}
=head2 each_DBLink
Title : each_DBLink
Usage : foreach $ref ( $self->each_DBlink() )
Function: gets an array of DBlink of objects
Example :
Returns :
Args :
=cut
sub each_DBLink{
my ($self) = @_;
return @{$self->{'link'}};
}
=head2 restriction_changes
Title : restriction_changes
Usage : $obj->restriction_changes();
Function:
Returns a string containing a list of restriction
enzyme changes of form +EcoRI, separated by
commas. Strings need to be valid restriction enzyme names
as stored in REBASE. allele_ori and allele_mut need to be assigned.
Example :
Returns : string
Args : string
=cut
sub restriction_changes {
my ($self) = @_;
if (not $self->{'re_changes'}) {
my %re = &_enzymes;
# complain if used on AA data
if ($self->isa('Bio::Variation::AAChange')) {
$self->throw('Restriction enzymes do not bite polypeptides!');
}
#sanity checks
$self->warn('Upstream sequence is empty!')
if $self->upStreamSeq eq '';
$self->warn('Downstream sequence is empty!')
if $self->dnStreamSeq eq '';
# $self->warn('Original allele sequence is empty!')
# if $self->allele_ori eq '';
# $self->warn('Mutated allele sequence is empty!')
# if $self->allele_mut eq '';
#reuse the non empty DNA level list at RNA level if the flanks are identical
#Hint: Check DNAMutation object first
if ($self->isa('Bio::Variation::RNAChange') and $self->DNAMutation and
$self->upStreamSeq eq $self->DNAMutation->upStreamSeq and
$self->dnStreamSeq eq $self->DNAMutation->dnStreamSeq and
$self->DNAMutation->restriction_changes ne '' ) {
$self->{'re_changes'} = $self->DNAMutation->restriction_changes;
} else {
#maximum length of a type II restriction site in the current REBASE
my ($le_dn) = 15;
my ($le_up) = $le_dn;
#reduce the flank lengths if the desired length is not available
$le_dn = CORE::length ($self->dnStreamSeq) if $le_dn > CORE::length ($self->dnStreamSeq);
$le_up = CORE::length ($self->upStreamSeq) if $le_up > CORE::length ($self->upStreamSeq);
#Build sequence strings to compare
my ($oriseq, $mutseq);
$oriseq = $mutseq = substr($self->upStreamSeq, -$le_up, $le_up);
$oriseq .= $self->allele_ori->seq if $self->allele_ori->seq;
$mutseq .= $self->allele_mut->seq if $self->allele_mut->seq;
$oriseq .= substr($self->dnStreamSeq, 0, $le_dn);
$mutseq .= substr($self->dnStreamSeq, 0, $le_dn);
# ... and their reverse complements
my $oriseq_rev = _revcompl ($oriseq);
my $mutseq_rev = _revcompl ($mutseq);
# collect results into a string
my $rec = '';
foreach my $enz (sort keys (%re)) {
my $site = $re{$enz};
my @ori = ($oriseq=~ /$site/g);
my @mut = ($mutseq=~ /$site/g);
my @ori_r = ($oriseq_rev =~ /$site/g);
my @mut_r = ($mutseq_rev =~ /$site/g);
$rec .= '+'. $enz. ", "
if (scalar @ori < scalar @mut) or (scalar @ori_r < scalar @mut_r);
$rec .= '-'. $enz. ", "
if (scalar @ori > scalar @mut) or (scalar @ori_r > scalar @mut_r);
}
$rec = substr($rec, 0, CORE::length($rec) - 2) if $rec ne '';
$self->{'re_changes'} = $rec;
}
}
return $self->{'re_changes'}
}
sub _revcompl {
# side effect: lower case letters
my ($seq) = shift;
$seq = lc $seq;
$seq =~ tr/acgtrymkswhbvdnx/tgcayrkmswdvbhnx/;
return CORE::reverse $seq;
}
sub _enzymes {
#REBASE version 005 type2.005
my %enzymes = (
'AarI' => 'cacctgc',
'AatII' => 'gacgtc',
'AccI' => 'gt[ac][gt]ac',
'AceIII' => 'cagctc',
'AciI' => 'ccgc',
'AclI' => 'aacgtt',
'AcyI' => 'g[ag]cg[ct]c',
'AflII' => 'cttaag',
'AflIII' => 'ac[ag][ct]gt',
'AgeI' => 'accggt',
'AhaIII' => 'tttaaa',
'AloI' => 'gaac[acgt][acgt][acgt][acgt][acgt][acgt]tcc',
'AluI' => 'agct',
'AlwNI' => 'cag[acgt][acgt][acgt]ctg',
'ApaBI' => 'gca[acgt][acgt][acgt][acgt][acgt]tgc',
'ApaI' => 'gggccc',
'ApaLI' => 'gtgcac',
'ApoI' => '[ag]aatt[ct]',
'AscI' => 'ggcgcgcc',
'AsuI' => 'gg[acgt]cc',
'AsuII' => 'ttcgaa',
'AvaI' => 'c[ct]cg[ag]g',
'AvaII' => 'gg[at]cc',
'AvaIII' => 'atgcat',
'AvrII' => 'cctagg',
'BaeI' => 'ac[acgt][acgt][acgt][acgt]gta[ct]c',
'BalI' => 'tggcca',
'BamHI' => 'ggatcc',
'BbvCI' => 'cctcagc',
'BbvI' => 'gcagc',
'BbvII' => 'gaagac',
'BccI' => 'ccatc',
'Bce83I' => 'cttgag',
'BcefI' => 'acggc',
'BcgI' => 'cga[acgt][acgt][acgt][acgt][acgt][acgt]tgc',
'BciVI' => 'gtatcc',
'BclI' => 'tgatca',
'BetI' => '[at]ccgg[at]',
'BfiI' => 'actggg',
'BglI' => 'gcc[acgt][acgt][acgt][acgt][acgt]ggc',
'BglII' => 'agatct',
'BinI' => 'ggatc',
'BmgI' => 'g[gt]gccc',
'BplI' => 'gag[acgt][acgt][acgt][acgt][acgt]ctc',
'Bpu10I' => 'cct[acgt]agc',
'BsaAI' => '[ct]acgt[ag]',
'BsaBI' => 'gat[acgt][acgt][acgt][acgt]atc',
'BsaXI' => 'ac[acgt][acgt][acgt][acgt][acgt]ctcc',
'BsbI' => 'caacac',
'BscGI' => 'cccgt',
'BseMII' => 'ctcag',
'BsePI' => 'gcgcgc',
'BseRI' => 'gaggag',
'BseSI' => 'g[gt]gc[ac]c',
'BsgI' => 'gtgcag',
'BsiI' => 'cacgag',
'BsiYI' => 'cc[acgt][acgt][acgt][acgt][acgt][acgt][acgt]gg',
'BsmAI' => 'gtctc',
'BsmI' => 'gaatgc',
'Bsp1407I' => 'tgtaca',
'Bsp24I' => 'gac[acgt][acgt][acgt][acgt][acgt][acgt]tgg',
'BspGI' => 'ctggac',
'BspHI' => 'tcatga',
'BspLU11I' => 'acatgt',
'BspMI' => 'acctgc',
'BspMII' => 'tccgga',
'BsrBI' => 'ccgctc',
'BsrDI' => 'gcaatg',
'BsrI' => 'actgg',
'BstEII' => 'ggt[acgt]acc',
'BstXI' => 'cca[acgt][acgt][acgt][acgt][acgt][acgt]tgg',
'BtrI' => 'cacgtc',
'BtsI' => 'gcagtg',
'Cac8I' => 'gc[acgt][acgt]gc',
'CauII' => 'cc[cg]gg',
'Cfr10I' => '[ag]ccgg[ct]',
'CfrI' => '[ct]ggcc[ag]',
'CjeI' => 'cca[acgt][acgt][acgt][acgt][acgt][acgt]gt',
'CjePI' => 'cca[acgt][acgt][acgt][acgt][acgt][acgt][acgt]tc',
'ClaI' => 'atcgat',
'CviJI' => '[ag]gc[ct]',
'CviRI' => 'tgca',
'DdeI' => 'ct[acgt]ag',
'DpnI' => 'gatc',
'DraII' => '[ag]gg[acgt]cc[ct]',
'DraIII' => 'cac[acgt][acgt][acgt]gtg',
'DrdI' => 'gac[acgt][acgt][acgt][acgt][acgt][acgt]gtc',
'DrdII' => 'gaacca',
'DsaI' => 'cc[ag][ct]gg',
'Eam1105I' => 'gac[acgt][acgt][acgt][acgt][acgt]gtc',
'EciI' => 'ggcgga',
'Eco31I' => 'ggtctc',
'Eco47III' => 'agcgct',
'Eco57I' => 'ctgaag',
'EcoNI' => 'cct[acgt][acgt][acgt][acgt][acgt]agg',
'EcoRI' => 'gaattc',
'EcoRII' => 'cc[at]gg',
'EcoRV' => 'gatatc',
'Esp3I' => 'cgtctc',
'EspI' => 'gct[acgt]agc',
'FauI' => 'cccgc',
'FinI' => 'gggac',
'Fnu4HI' => 'gc[acgt]gc',
'FnuDII' => 'cgcg',
'FokI' => 'ggatg',
'FseI' => 'ggccggcc',
'GdiII' => 'cggcc[ag]',
'GsuI' => 'ctggag',
'HaeI' => '[at]ggcc[at]',
'HaeII' => '[ag]gcgc[ct]',
'HaeIII' => 'ggcc',
'HaeIV' => 'ga[ct][acgt][acgt][acgt][acgt][acgt][ag]tc',
'HgaI' => 'gacgc',
'HgiAI' => 'g[at]gc[at]c',
'HgiCI' => 'gg[ct][ag]cc',
'HgiEII' => 'acc[acgt][acgt][acgt][acgt][acgt][acgt]ggt',
'HgiJII' => 'g[ag]gc[ct]c',
'HhaI' => 'gcgc',
'Hin4I' => 'ga[cgt][acgt][acgt][acgt][acgt][acgt][acg]tc',
'HindII' => 'gt[ct][ag]ac',
'HindIII' => 'aagctt',
'HinfI' => 'ga[acgt]tc',
'HpaI' => 'gttaac',
'HpaII' => 'ccgg',
'HphI' => 'ggtga',
'Hpy178III' => 'tc[acgt][acgt]ga',
'Hpy188I' => 'tc[acgt]ga',
'Hpy99I' => 'cg[at]cg',
'KpnI' => 'ggtacc',
'Ksp632I' => 'ctcttc',
'MaeI' => 'ctag',
'MaeII' => 'acgt',
'MaeIII' => 'gt[acgt]ac',
'MboI' => 'gatc',
'MboII' => 'gaaga',
'McrI' => 'cg[ag][ct]cg',
'MfeI' => 'caattg',
'MjaIV' => 'gt[acgt][acgt]ac',
'MluI' => 'acgcgt',
'MmeI' => 'tcc[ag]ac',
'MnlI' => 'cctc',
'MseI' => 'ttaa',
'MslI' => 'ca[ct][acgt][acgt][acgt][acgt][ag]tg',
'MstI' => 'tgcgca',
'MwoI' => 'gc[acgt][acgt][acgt][acgt][acgt][acgt][acgt]gc',
'NaeI' => 'gccggc',
'NarI' => 'ggcgcc',
'NcoI' => 'ccatgg',
'NdeI' => 'catatg',
'NheI' => 'gctagc',
'NlaIII' => 'catg',
'NlaIV' => 'gg[acgt][acgt]cc',
'NotI' => 'gcggccgc',
'NruI' => 'tcgcga',
'NspBII' => 'c[ac]gc[gt]g',
'NspI' => '[ag]catg[ct]',
'PacI' => 'ttaattaa',
'Pfl1108I' => 'tcgtag',
'PflMI' => 'cca[acgt][acgt][acgt][acgt][acgt]tgg',
'PleI' => 'gagtc',
'PmaCI' => 'cacgtg',
'PmeI' => 'gtttaaac',
'PpiI' => 'gaac[acgt][acgt][acgt][acgt][acgt]ctc',
'PpuMI' => '[ag]gg[at]cc[ct]',
'PshAI' => 'gac[acgt][acgt][acgt][acgt]gtc',
'PsiI' => 'ttataa',
'PstI' => 'ctgcag',
'PvuI' => 'cgatcg',
'PvuII' => 'cagctg',
'RleAI' => 'cccaca',
'RsaI' => 'gtac',
'RsrII' => 'cgg[at]ccg',
'SacI' => 'gagctc',
'SacII' => 'ccgcgg',
'SalI' => 'gtcgac',
'SanDI' => 'ggg[at]ccc',
'SapI' => 'gctcttc',
'SauI' => 'cct[acgt]agg',
'ScaI' => 'agtact',
'ScrFI' => 'cc[acgt]gg',
'SduI' => 'g[agt]gc[act]c',
'SecI' => 'cc[acgt][acgt]gg',
'SexAI' => 'acc[at]ggt',
'SfaNI' => 'gcatc',
'SfeI' => 'ct[ag][ct]ag',
'SfiI' => 'ggcc[acgt][acgt][acgt][acgt][acgt]ggcc',
'SgfI' => 'gcgatcgc',
'SgrAI' => 'c[ag]ccgg[ct]g',
'SimI' => 'gggtc',
'SmaI' => 'cccggg',
'SmlI' => 'ct[ct][ag]ag',
'SnaBI' => 'tacgta',
'SnaI' => 'gtatac',
'SpeI' => 'actagt',
'SphI' => 'gcatgc',
'SplI' => 'cgtacg',
'SrfI' => 'gcccgggc',
'Sse232I' => 'cgccggcg',
'Sse8387I' => 'cctgcagg',
'Sse8647I' => 'agg[at]cct',
'SspI' => 'aatatt',
'Sth132I' => 'cccg',
'StuI' => 'aggcct',
'StyI' => 'cc[at][at]gg',
'SwaI' => 'atttaaat',
'TaqI' => 'tcga',
'TaqII' => 'gaccga',
'TatI' => '[at]gtac[at]',
'TauI' => 'gc[cg]gc',
'TfiI' => 'ga[at]tc',
'TseI' => 'gc[at]gc',
'Tsp45I' => 'gt[cg]ac',
'Tsp4CI' => 'ac[acgt]gt',
'TspEI' => 'aatt',
'TspRI' => 'ca[cg]tg[acgt][acgt]',
'Tth111I' => 'gac[acgt][acgt][acgt]gtc',
'Tth111II' => 'caa[ag]ca',
'UbaGI' => 'cac[acgt][acgt][acgt][acgt]gtg',
'UbaPI' => 'cgaacg',
'VspI' => 'attaat',
'XbaI' => 'tctaga',
'XcmI' => 'cca[acgt][acgt][acgt][acgt][acgt][acgt][acgt][acgt][acgt]tgg',
'XhoI' => 'ctcgag',
'XhoII' => '[ag]gatc[ct]',
'XmaIII' => 'cggccg',
'XmnI' => 'gaa[acgt][acgt][acgt][acgt]ttc'
);
return %enzymes;
}
1;