Raw content of Bio::Variation::VariantI # $Id: VariantI.pm,v 1.12 2002/10/22 07:38:49 lapp Exp $ # # BioPerl module for Bio::Variation::VariantI # # Cared for by Heikki Lehvaslaiho <heikki@ebi.ac.uk> # # 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://bio.perl.org/MailList.html - About the mailing lists =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 email or the web: bioperl-bugs@bio.perl.org http://bugzilla.bioperl.org/ =head1 AUTHOR - Heikki Lehvaslaiho Email: heikki@ebi.ac.uk Address: EMBL Outstation, European Bioinformatics Institute Wellcome Trust Genome Campus, Hinxton Cambs. CB10 1SD, United Kingdom =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; $VERSION=1.0; use vars qw(@ISA); use strict; use Bio::Root::Root; use Bio::DBLinkContainerI; # Object preamble - inheritance use Bio::SeqFeature::Generic; @ISA = 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) = @_; $self->throw("[$self] has not implemeted method 'label'"); } =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 (undef); } else { $self->{'seqDiff'} = $value; } } unless (exists $self->{'seqDiff'}) { return (undef); } 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;