LOCUS       sequence-211626-       16700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one genome editing vector in M. polymorpha (hygromycin 
            resistant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16700)
  AUTHORS   Sugano SS, Nishihama R, Shirakawa M, Takagi J, Matsuda Y, Ishida S, 
            Shimada T, Hara-Nishimura I, Osakabe K, Kohchi T
  TITLE     Efficient CRISPR/Cas9-based genome editing and its application to 
            conditional genetic analysis in Marchantia polymorpha.
  JOURNAL   PLoS One. 2018 Oct 31;13(10):e0205117. doi: 
            10.1371/journal.pone.0205117. eCollection 2018.
  PUBMED    30379827
REFERENCE   2  (bases 1 to 16700)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2018-10-31"; volume: "13(10):e0205117. doi"; pages: " 
            10.1371/journal.pone.0205117. eCollection 2018"
COMMENT     Sequence Label: pMpGE013
FEATURES             Location/Qualifiers
     source          1..16700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1743..1767
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1788..5891
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1788..5891
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5904..5924
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(6427..6451)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    6480..6504
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_RNA        7065..7140
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(7182..7206)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     terminator      7246..7498
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(7531..7547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7531..7547)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7544..7566)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7555..7571
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7579..7585,7586..7603,7604..7609))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7624..7645
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    7730..7904
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     CDS             complement(7945..8970)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRSQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     K"
     promoter        complement(9037..9713)
                     /label=CaMV 35S promoter (enhanced)
                     /note="cauliflower mosaic virus 35S promoter with a 
                     duplicated enhancer region"
     primer_bind     complement(9080..9100)
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     misc_feature    9908..9932
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     CDS             10453..11244
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      11490..12078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11979..11998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12232..12249
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    12264..12404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(12330..12349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12490..12512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     rep_origin      12748..12942
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(13008..14081)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     CDS             complement(14510..15139)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    16440..16464
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     primer_bind     16652..16674
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     16666..16683
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     16667..16683
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 caaatgagtc acacacattg ttgagagaca tatcaaaata actcaattgt aaggtaagtt
       61 gatagatttc ccagttaaat gcattatttt aacaatattt gtatctcaaa ttttcaaaat
      121 catatcaaat ttgcagatct tgatgatgat tcacaactct gtcttcgatt tttgattgtt
      181 aacgggtatc gatatttggg taacttaaaa agaatttgct aaagatctta aaaaacagat
      241 taaaaaaacc tttcaaaata gctaatcctt taaatccgtt tcacaatata atgctcattg
      301 atgataatcg aaaagattcc attttaaagt gcatataaat tgcttaatta tctctttctg
      361 tctcgtaata ttttgttctc tcctattgga tcggacccgc atcttctcga tcagttcact
      421 atactaatat ttggtcaact tcaagagaga ttagaacttc gttctccaaa gtgtaaaggc
      481 cgacaagcag aaaaggaaac ttcaatcaga agccttctag gttttcccgc gtcccaaaaa
      541 gcgaagggag gacggggagg acggtgaggc cagaagaaga aagcaacagt aaaatctcat
      601 gtgattgcgt gcatgtcatg gatctatggg atgctagtcc accgtttgga agtatgtgca
      661 gaaggagaaa ggcgcgggag gccaaaagcg acttcggcca cctctctact ttctagaaag
      721 tttgctcgca gcttgtaata agcgcggaaa aggtactcga aaaaccggag caatcggtgt
      781 ggtatcagca gtcgggttcg ctaaacctac gtcttcttat tttggaatgt taaattgaaa
      841 gggcgtgccg gccctaagca cggacgatgt gacagacgga ggagtgtgag caattcggat
      901 gacgatgctg gccggcagtc acgacaaaga gtccgaaggg gcatggacag ggcaatatat
      961 aaatggaaaa gaggctttga atattcctaa cccttcgtcg tcggcctccc aacttgccct
     1021 cgcatgctga attggccgcc tatctgcgaa tggccggccg ccgaccctaa gtcgcaataa
     1081 cttgaacgga gccgatgcaa agtccttatc gaacgctcgt tattataaac ggggcagacc
     1141 taggtattta attatatcaa aaccctccgc aatttcgcta atcattccct aatttcttct
     1201 actttggcgt ctagtgctgt ggtcgcgctt gctctcaaag ctcgctggga ggcagtggag
     1261 cgtctggctt aaaatcgatc gccgcaaccc tagagttcct ttcacttctt tctctcttgc
     1321 gtggagagca tcgacatcga agagctgcat cttgtcttct ggtatgcgcc ttgtgccctc
     1381 gtctctgtac gtcgttacga ttgctcgata ttgtgattgt ctagacctga aacccgactg
     1441 gagcttttac gtggtgtcga ttggggcctc gagcgagtga ttttttaggt tcgttttagc
     1501 caagtacatc ccactttggt cagtcctgtt atctcggtaa ggtgttcagt tcggtctctg
     1561 gtgcggaggt gcctcgtaag tttcctgcat gatagatttg tctatccttg acttgaagag
     1621 gagtcgaagg tcagcgaggt tggctgtgtg ctatggacgt cggtgctcct tggttccgtg
     1681 cttctatggg tgttgcctca acagtattga tgtgcctgca gaaaggttga ctagagttat
     1741 caacaagttt gtacaaaaaa gcaggctccg cggccgcccc cttcaccatg gataagaagt
     1801 actctatcgg actcgatatc ggaactaact ctgtgggatg ggctgtgatc accgatgagt
     1861 acaaggtgcc atctaagaag ttcaaggttc tcggaaacac cgataggcac tctatcaaga
     1921 aaaaccttat cggtgctctc ctcttcgatt ctggtgaaac tgctgaggct accagactca
     1981 agagaaccgc tagaagaagg tacaccagaa gaaagaacag gatctgctac ctccaagaga
     2041 tcttctctaa cgagatggct aaagtggatg attcattctt ccacaggctc gaagagtcat
     2101 tcctcgtgga agaagataag aagcacgaga ggcaccctat cttcggaaac atcgttgatg
     2161 aggtggcata ccacgagaag taccctacta tctaccacct cagaaagaag ctcgttgatt
     2221 ctactgataa ggctgatctc aggctcatct acctcgctct cgctcacatg atcaagttca
     2281 gaggacactt cctcatcgag ggtgatctca accctgataa ctctgatgtg gataagttgt
     2341 tcatccagct cgtgcagacc tacaaccagc ttttcgaaga gaaccctatc aacgcttcag
     2401 gtgtggatgc taaggctatc ctctctgcta ggctctctaa gtcaagaagg cttgagaacc
     2461 tcattgctca gctccctggt gagaagaaga acggactttt cggaaacttg atcgctctct
     2521 ctctcggact cacccctaac ttcaagtcta acttcgatct cgctgaggat gcaaagctcc
     2581 agctctcaaa ggatacctac gatgatgatc tcgataacct cctcgctcag atcggagatc
     2641 agtacgctga tttgttcctc gctgctaaga acctctctga tgctatcctc ctcagtgata
     2701 tcctcagagt gaacaccgag atcaccaagg ctccactctc agcttctatg atcaagagat
     2761 acgatgagca ccaccaggat ctcacacttc tcaaggctct tgttagacag cagctcccag
     2821 agaagtacaa agagattttc ttcgatcagt ctaagaacgg atacgctggt tacatcgatg
     2881 gtggtgcatc tcaagaagag ttctacaagt tcatcaagcc tatcctcgag aagatggatg
     2941 gaaccgagga actcctcgtg aagctcaata gagaggatct tctcagaaag cagaggacct
     3001 tcgataacgg atctatccct catcagatcc acctcggaga gttgcacgct atccttagaa
     3061 ggcaagagga tttctaccca ttcctcaagg ataacaggga aaagattgag aagattctca
     3121 ccttcagaat cccttactac gtgggacctc tcgctagagg aaactcaaga ttcgcttgga
     3181 tgaccagaaa gtctgaggaa accatcaccc cttggaactt cgaagaggtg gtggataagg
     3241 gtgctagtgc tcagtctttc atcgagagga tgaccaactt cgataagaac cttccaaacg
     3301 agaaggtgct ccctaagcac tctttgctct acgagtactt caccgtgtac aacgagttga
     3361 ccaaggttaa gtacgtgacc gagggaatga ggaagcctgc ttttttgtca ggtgagcaaa
     3421 agaaggctat cgttgatctc ttgttcaaga ccaacagaaa ggtgaccgtg aagcagctca
     3481 aagaggatta cttcaagaaa atcgagtgct tcgattcagt tgagatttct ggtgttgagg
     3541 ataggttcaa cgcatctctc ggaacctacc acgatctcct caagatcatt aaggataagg
     3601 atttcttgga taacgaggaa aacgaggata tcttggagga tatcgttctt accctcaccc
     3661 tctttgaaga tagagagatg attgaagaaa ggctcaagac ctacgctcat ctcttcgatg
     3721 ataaggtgat gaagcagttg aagagaagaa gatacactgg ttggggaagg ctctcaagaa
     3781 agctcattaa cggaatcagg gataagcagt ctggaaagac aatccttgat ttcctcaagt
     3841 ctgatggatt cgctaacaga aacttcatgc agctcatcca cgatgattct ctcaccttta
     3901 aagaggatat ccagaaggct caggtttcag gacagggtga tagtctccat gagcatatcg
     3961 ctaacctcgc tggatctcct gcaatcaaga agggaatcct ccagactgtg aaggttgtgg
     4021 atgagttggt gaaggtgatg ggaaggcata agcctgagaa catcgtgatc gaaatggcta
     4081 gagagaacca gaccactcag aagggacaga agaactctag ggaaaggatg aagaggatcg
     4141 aggaaggtat caaagagctt ggatctcaga tcctcaaaga gcaccctgtt gagaacactc
     4201 agctccagaa tgagaagctc tacctctact acctccagaa cggaagggat atgtatgtgg
     4261 atcaagagtt ggatatcaac aggctctctg attacgatgt tgatcatatc gtgccacagt
     4321 cattcttgaa ggatgattct atcgataaca aggtgctcac caggtctgat aagaacaggg
     4381 gtaagagtga taacgtgcca agtgaagagg ttgtgaagaa aatgaagaac tattggaggc
     4441 agctcctcaa cgctaagctc atcactcaga gaaagttcga taacttgact aaggctgaga
     4501 ggggaggact ctctgaattg gataaggcag gattcatcaa gaggcagctt gtggaaacca
     4561 ggcagatcac taagcacgtt gcacagatcc tcgattctag gatgaacacc aagtacgatg
     4621 agaacgataa gttgatcagg gaagtgaagg ttatcaccct caagtcaaag ctcgtgtctg
     4681 atttcagaaa ggatttccaa ttctacaagg tgagggaaat caacaactac caccacgctc
     4741 acgatgctta ccttaacgct gttgttggaa ccgctctcat caagaagtat cctaagctcg
     4801 agtcagagtt cgtgtacggt gattacaagg tgtacgatgt gaggaagatg atcgctaagt
     4861 ctgagcaaga gatcggaaag gctaccgcta agtatttctt ctactctaac atcatgaatt
     4921 tcttcaagac cgagattacc ctcgctaacg gtgagatcag aaagaggcca ctcatcgaga
     4981 caaacggtga aacaggtgag atcgtgtggg ataagggaag ggatttcgct accgttagaa
     5041 aggtgctctc tatgccacag gtgaacatcg ttaagaaaac cgaggtgcag accggtggat
     5101 tctctaaaga gtctatcctc cctaagagga actctgataa gctcattgct aggaagaagg
     5161 attgggaccc taagaaatac ggtggtttcg attctcctac cgtggcttac tctgttctcg
     5221 ttgtggctaa ggttgagaag ggaaagagta agaagctcaa gtctgttaag gaacttctcg
     5281 gaatcactat catggaaagg tcatctttcg agaagaaccc aatcgatttc ctcgaggcta
     5341 agggatacaa agaggttaag aaggatctca tcatcaagct cccaaagtac tcactcttcg
     5401 aactcgagaa cggtagaaag aggatgctcg cttctgctgg tgagcttcaa aagggaaacg
     5461 agcttgctct cccatctaag tacgttaact ttctttacct cgcttctcac tacgagaagt
     5521 tgaagggatc tccagaagat aacgagcaga agcaactttt cgttgagcag cacaagcact
     5581 acttggatga gatcatcgag cagatctctg agttctctaa aagggtgatc ctcgctgatg
     5641 caaacctcga taaggtgttg tctgcttaca acaagcacag agataagcct atcagggaac
     5701 aggcagagaa catcatccat ctcttcaccc ttaccaacct cggtgctcct gctgctttca
     5761 agtacttcga tacaaccatc gataggaaga gatacacctc taccaaagaa gtgctcgatg
     5821 ctaccctcat ccatcagtct atcactggac tctacgagac taggatcgat ctctcacagc
     5881 tcggtggtga ttcaagggct gatcctaaga agaagaggaa ggtttgaggc gcgccgagct
     5941 ccaggcctcc cagctttcgt ccgtatcatc ggtttcgaca acgttcgtca agttcaatgc
     6001 atcagtttca ttgcccacac accagaatcc tactaagttt gagtattatg gcattggaaa
     6061 agctgttttc ttctatcatt tgttctgctt gtaatttact gtgttctttc agtttttgtt
     6121 ttcggacatc aaaatgcaaa tggatggata agagttaata aatgatatgg tccttttgtt
     6181 cattctcaaa ttattattat ctgttgtttt tactttaatg ggttgaattt aagtaagaaa
     6241 ggaactaaca gtgtgatatt aaggtgcaat gttagacata taaaacagtc tttcacctct
     6301 ctttggttat gtcttgaatt ggtttgtttc ttcacttatc tgtgtaatca agtttactat
     6361 gagtctatga tcaagtaatt atgcaatcaa gttaagtaca gtataggctt aagggtgggc
     6421 gcgccgaccc agctttcttg tacaaagtgg ttgataacag cggttgacta gagttatcaa
     6481 caagtttgta caaaaaagca ggctccgcgg ccgccccctt gtaacgtgag actactacac
     6541 tcctcaacgc aagttctatc tcatatctct gcaagagctt attccaagaa actttcttgt
     6601 gcttctatgc atgcccaggt accttaagtc gcgcgcatag tcacttcaca gccttttcag
     6661 ttctcccttt gatggctcag agtgacgcaa ctgcacaaaa ataagaaaaa agtcgaagcg
     6721 tcacgagaga tgtatccggc tagtttcctt ggagttcgga ttgctctctt tctctgtctc
     6781 cccgaaccat atggaattta tgaactgctg ttcactagtc agtcacttgt ttcagaactt
     6841 ttgagtggtc gatttatcca ccttcttgtt tcttcctggg tccagaatct gacttcttga
     6901 gacaacaaac agtgcacagt ggatcgaaga atgaccacag ttgagacacg tggcgctggc
     6961 ctcatttatt gcaccatacg tttagtgaag aactgaataa tggtggatat aaagcagagt
     7021 tgcacccagc ctctcgaaat gcaggtgatg actcacctgc ataagtttta gagctagaaa
     7081 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     7141 tttttttcta gtatcgggaa ttctaaaggg tgggcgcgcc gacccagctt tcttgtacaa
     7201 agtggttgat aacagcgctt agaggcttag agctcgaatt tccccgatcg ttcaaacatt
     7261 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa
     7321 tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg
     7381 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa
     7441 atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg
     7501 gaattggttc cggaaccaat tcgtaatcat gtcatagctg tttcctgtgt gaaattgtta
     7561 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     7621 ctaatgagtg agctaactca cattaggctg aattaggcgc gcctatttct gaattcaaca
     7681 cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg atctggattt
     7741 tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat tttacaaata
     7801 caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct ataggaaccc
     7861 taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag cttgtcgatc
     7921 gacagatccc ggtcggcatc tactctattt ctttgccctc ggacgagtgc tggggcgtcg
     7981 gtttccacta tcggcgagta cttctacaca gccatcggtc cagacggccg cgcttctgcg
     8041 ggcgatttgt gtacgcccga cagtcccggc tccggatcgg acgattgcgt cgcatcgacc
     8101 ctgcgcccaa gctgcatcat cgaaattgcc gtcaaccaag ctctgataga gttggtcaag
     8161 accaatgcgg agcatatacg cccggagtcg tggcgatcct gcaagctccg gatgcctccg
     8221 ctcgaagtag cgcgtctgct gctccataca agccaaccac ggcctccaga agaagatgtt
     8281 ggcgacctcg tattgggaat ccccgaacat cgcctcgctc cagtcaatga ccgctgttat
     8341 gcggccattg tccgtcagga cattgttgga gccgaaatcc gcgtgcacga ggtgccggac
     8401 ttcggggcag tcctcggccc aaagcatcag ctcatcgaga gcctgcgcga cggacgcact
     8461 gacggtgtcg tccatcacag tttgccagtg atacacatgg ggatcagcaa tcgcgcatat
     8521 gaaatcacgc catgtagtgt attgaccgat tccttgcggt ccgaatgggc cgaacccgct
     8581 cgtctggcta agatcggccg cagcgatcgc atccatagcc tccgcgaccg gttgtagaac
     8641 agcgggcagt tcggtttcag gcaggtcttg caacgtgaca ccctgtgaac ggcgggagat
     8701 gcaataggtc aggctctcgc taaactcccc aatgtcaagc acttccggaa tcgggagcgc
     8761 ggccgatgca aagtgccgat aaacataacg atctttgtag aaaccatcgg cgcagctatt
     8821 tacccgcagg acatatccac gccctcctac atcgaagctg aaagcacgag attcttcgcc
     8881 ctccgagagc tgcatcaggt cggagacgct gtcgaacttt tcgatcagaa acttctcgac
     8941 agacgtcgcg gtgagttcag gctttttcat atctcattgc cccccggatc tgcgaaagct
     9001 cgagagagat agatttgtag agagagactg gtgatttcag cgtgtcctct ccaaatgaaa
     9061 tgaacttcct tatatagagg aagggtcttg cgaaggatag tgggattgtg cgtcatccct
     9121 tacgtcagtg gagatatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc
     9181 tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc
     9241 atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc caccttcctt
     9301 ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga ggaggtttcc
     9361 cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga ctgtatcttt
     9421 gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttcacatcaa tccacttgct
     9481 ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat
     9541 ctttgggacc actgtcggca gaggcatctt gaacgatagc ctttccttta tcgcaatgat
     9601 ggcatttgta ggtgccacct tccttttcta ctgtcctttt gatgaagtga cagatagctg
     9661 ggcaatggaa tccgaggagg tttcccgata ttaccctttg ttgaaaagtc tcaatagccc
     9721 tttggtcttc tgagactgta tctttgatat tcttggagta gacgagagtg tcgtgctcca
     9781 ccatgttggc aagctgctct agccaatacg caaaccgaat tcagaaataa attcagccta
     9841 attcggcgtt aattcagtac attaaaaacg tccgcaatgt gttattaagt tgtctaagcg
     9901 tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc cccgaccggc
     9961 agctcggcac aaaatcacca ctcgatacag gcagcccatc agtccgggac ggcgtcagcg
    10021 ggagagccgt tgtaaggcgg cagactttgc tcatgttacc gatgctattc ggaagaacgg
    10081 caactaagct gccgggtttg aaacacggat gatctcgcgg agggtagcat gttgattgta
    10141 acgatgacag agcgttgctg cctgtgatca attcgggcac gaacccagtg gacataagcc
    10201 tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc
    10261 ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt
    10321 ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt
    10381 cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca
    10441 aagttaaaca tcatggggga agcggtgatc gccgaagtat cgactcaact atcagaggta
    10501 gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc
    10561 gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta
    10621 aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc
    10681 cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc
    10741 attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac
    10801 attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca
    10861 aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg
    10921 gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg
    10981 cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc
    11041 gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg
    11101 ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa
    11161 gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag
    11221 atcaccaagg tagtcggcaa ataatgtcta gctagaaatt cgttcaagcc gacgccgctt
    11281 cgcggcgcgg cttaactcaa gcgttagatg cactaagcac ataattgctc acagccaaac
    11341 tatcaggtca agtctgcttt tattattttt aagcgtgcat aataagccct acacaaattg
    11401 ggagatatat catgcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
    11461 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
    11521 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
    11581 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
    11641 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
    11701 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
    11761 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
    11821 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
    11881 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
    11941 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
    12001 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
    12061 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
    12121 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
    12181 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
    12241 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
    12301 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
    12361 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
    12421 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
    12481 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
    12541 ggcagggtgc cttgatgtgg gcgccggcgg tcgagtggcg acggcgcggc ttgtccgcgc
    12601 cctggtagat tgcctggccg taggccagcc atttttgagc ggccagcggc cgcgataggc
    12661 cgacgcgaag cggcggggcg tagggagcgc agcgaccgaa gggtaggcgc tttttgcagc
    12721 tcttcggctg tgcgctggcc agacagttat gcacaggcca ggcgggtttt aagagtttta
    12781 ataagtttta aagagtttta ggcggaaaaa tcgccttttt tctcttttat atcagtcact
    12841 tacatgtgtg accggttccc aatgtacggc tttgggttcc caatgtacgg gttccggttc
    12901 ccaatgtacg gctttgggtt cccaatgtac gtgctatcca caggaaagag accttttcga
    12961 cctttttccc ctgctagggc aatttgccct agcatctgct ccgtacatta ggaaccggcg
    13021 gatgcttcgc cctcgatcag gttgcggtag cgcatgacta ggatcgggcc agcctgcccc
    13081 gcctcctcct tcaaatcgta ctccggcagg tcatttgacc cgatcagctt gcgcacggtg
    13141 aaacagaact tcttgaactc tccggcgctg ccactgcgtt cgtagatcgt cttgaacaac
    13201 catctggctt ctgccttgcc tgcggcgcgg cgtgccaggc ggtagagaaa acggccgatg
    13261 ccgggatcga tcaaaaagta atcggggtga accgtcagca cgtccgggtt cttgccttct
    13321 gtgatctcgc ggtacatcca atcagctagc tcgatctcga tgtactccgg ccgcccggtt
    13381 tcgctcttta cgatcttgta gcggctaatc aaggcttcac cctcggatac cgtcaccagg
    13441 cggccgttct tggccttctt cgtacgctgc atggcaacgt gcgtggtgtt taaccgaatg
    13501 caggtttcta ccaggtcgtc tttctgcttt ccgccatcgg ctcgccggca gaacttgagt
    13561 acgtccgcaa cgtgtggacg gaacacgcgg ccgggcttgt ctcccttccc ttcccggtat
    13621 cggttcatgg attcggttag atgggaaacc gccatcagta ccaggtcgta atcccacaca
    13681 ctggccatgc cggccggccc tgcggaaacc tctacgtgcc cgtctggaag ctcgtagcgg
    13741 atcacctcgc cagctcgtcg gtcacgcttc gacagacgga aaacggccac gtccatgatg
    13801 ctgcgactat cgcgggtgcc cacgtcatag agcatcggaa cgaaaaaatc tggttgctcg
    13861 tcgcccttgg gcggcttcct aatcgacggc gcaccggctg ccggcggttg ccgggattct
    13921 ttgcggattc gatcagcggc cgcttgccac gattcaccgg ggcgtgcttc tgcctcgatg
    13981 cgttgccgct gggcggcctg cgcggccttc aacttctcca ccaggtcatc acccagcgcc
    14041 gcgccgattt gtaccgggcc ggatggtttg cgaccgctca cgccgattcc tcgggcttgg
    14101 gggttccagt gccattgcag ggccggcaga caacccagcc gcttacgcct ggccaaccgc
    14161 ccgttcctcc acacatgggg cattccacgg cgtcggtgcc tggttgttct tgattttcca
    14221 tgccgcctcc tttagccgct aaaattcatc tactcattta ttcatttgct catttactct
    14281 ggtagctgcg cgatgtattc agatagcagc tcggtaatgg tcttgccttg gcgtaccgcg
    14341 tacatcttca gcttggtgtg atcctccgcc ggcaactgaa agttgacccg cttcatggct
    14401 ggcgtgtctg ccaggctggc caacgttgca gccttgctgc tgcgtgcgct cggacggccg
    14461 gcacttagcg tgtttgtgct tttgctcatt ttctctttac ctcattaact caaatgagtt
    14521 ttgatttaat ttcagcggcc agcgcctgga cctcgcgggc agcgtcgccc tcgggttctg
    14581 attcaagaac ggttgtgccg gcggcggcag tgcctgggta gctcacgcgc tgcgtgatac
    14641 gggactcaag aatgggcagc tcgtacccgg ccagcgcctc ggcaacctca ccgccgatgc
    14701 gcgtgccttt gatcgcccgc gacacgacaa aggccgcttg tagccttcca tccgtgacct
    14761 caatgcgctg cttaaccagc tccaccaggt cggcggtggc ccatatgtcg taagggcttg
    14821 gctgcaccgg aatcagcacg aagtcggctg ccttgatcgc ggacacagcc aagtccgccg
    14881 cctggggcgc tccgtcgatc actacgaagt cgcgccggcc gatggccttc acgtcgcggt
    14941 caatcgtcgg gcggtcgatg ccgacaacgg ttagcggttg atcttcccgc acggccgccc
    15001 aatcgcgggc actgccctgg ggatcggaat cgactaacag aacatcggcc ccggcgagtt
    15061 gcagggcgcg ggctagatgg gttgcgatgg tcgtcttgcc tgacccgcct ttctggttaa
    15121 gtacagcgat aaccttcatg cgttcccctt gcgtatttgt ttatttactc atcgcatcat
    15181 atacgcagcg accgcatgac gcaagctgtt ttactcaaat acacatcacc tttttagacg
    15241 gcggcgctcg gtttcttcag cggccaagct ggccggccag gccgccagct tggcatcaga
    15301 caaaccggcc aggatttcat gcagccgcac ggttgagacg tgcgcgggcg gctcgaacac
    15361 gtacccggcc gcgatcatct ccgcctcgat ctcttcggta atgaaaaacg gttcgtcctg
    15421 gccgtcctgg tgcggtttca tgcttgttcc tcttggcgtt cattctcggc ggccgccagg
    15481 gcgtcggcct cggtcaatgc gtcctcacgg aaggcaccgc gccgcctggc ctcggtgggc
    15541 gtcacttcct cgctgcgctc aagtgcgcgg tacagggtcg agcgatgcac gccaagcagt
    15601 gcagccgcct ctttcacggt gcggccttcc tggtcgatca gctcgcgggc gtgcgcgatc
    15661 tgtgccgggg tgagggtagg gcgggggcca aacttcacgc ctcgggcctt ggcggcctcg
    15721 cgcccgctcc gggtgcggtc gatgattagg gaacgctcga actcggcaat gccggcgaac
    15781 acggtcaaca ccatgcggcc ggccggcgtg gtggtgtcgg cccacggctc tgccaggcta
    15841 cgcaggcccg cgccggcctc ctggatgcgc tcggcaatgt ccagtaggtc gcgggtgctg
    15901 cgggccaggc ggtctagcct ggtcactgtc acaacgtcgc cagggcgtag gtggtcaagc
    15961 atcctggcca gctccgggcg gtcgcgcctg gtgccggtga tcttctcgga aaacagcttg
    16021 gtgcagccgg ccgcgtgcag ttcggcccgt tggttggtca agtcctggtc gtcggtgctg
    16081 acgcgggcat agcccagcag gccagcggcg gcgctcttgt tcatggcgta atgtctccgg
    16141 ttctagtcgc aagtattcta ctttatgcga ctaaaacacg cgacaagaaa acgccaggaa
    16201 aagggcaggg cggcagcctg tcgcgtaact taggacttgt gcgacatgtc gttttcagaa
    16261 gacggctgca ctgaacgtca gaagccgact gcactatagc agcggagggg ttggatcaaa
    16321 gtactttgat cccgagggga accctgtggt tggcatgcac atacaaatgg acgaacggat
    16381 aaaccttttc acgccctttt aaatatccga ttattctaat aaacgctctt ttctcttagg
    16441 tttacccgcc aatatatcct gtcaaacact gatagtttaa actgaaggcg ggaaacgaca
    16501 atctgatcca agctcaagct gctctagcat tcgccattca ggctgcgcaa ctgttgggaa
    16561 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
    16621 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
    16681 agtgccaagc ttggcccggg
//
