LOCUS       sequence-247305-       10990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9-NLS 17 amino acid linker to monoavidin (SpCas9-NLS-MAV).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10990)
  AUTHORS   Roche PJR, Gytz H, Hussain F, Cameron CJF, Paquette D, Blanchette M,
            Dostie J, Nagar B, Akavia UD
  TITLE     Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair
            in Combination with Cas9 Monoavidin in Mammalian Cells.
  JOURNAL   CRISPR J. 2018 Dec;1:414-430. doi: 10.1089/crispr.2018.0045.
  PUBMED    31021244
REFERENCE   2  (bases 1 to 10990)
  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: "CRISPR J.";
            date: "2018-12"; volume: "1:414-430. doi"; pages: " 
            10.1089/crispr.2018.0045"
COMMENT     Sequence Label: spCas9-NLS-MAV
FEATURES             Location/Qualifiers
     source          1..10990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    5..144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     230..252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(246..437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1246..2328)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     2290..2309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2329..2406)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2612..2631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2715..2734
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2715..2733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2734..2758
                     /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)."
     RBS             2773..2795
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2806..2823
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2827..3924
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     3898..3921
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3955..3975
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3985..8088
                     /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             3985..8088
                     /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             8095..8115
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8122..8142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8194..8535
                     /codon_start=1
                     /product="monomeric streptavidin/rhizavidin hybrid (Lim et 
                     al., 2013)"
                     /label=mSA
                     /translation="AEAGITGTWYNQSGSTFTVTAGADGNLTGQYENRAQGTGCQNSPY
                     TLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATE
                     QGQDTFTKVK"
     CDS             8569..8586
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8639..8657)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      8653..8700
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      8737..9192
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8824..8843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9034..9055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(9284..10099)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(9284..10099)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     10007..10026
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     10091..10110
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      10221..10809
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10710..10729
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10963..10980
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
      541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
      601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2761 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcaccatcac
     2821 catggaaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
     2881 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
     2941 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
     3001 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
     3061 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
     3121 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
     3181 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
     3241 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
     3301 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
     3361 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
     3421 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
     3481 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
     3541 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
     3601 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag
     3661 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     3721 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc
     3781 gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     3841 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     3901 gaagccctga aagacgcgca gactaattcg agctcgaaca acaacaacac tagtgaaaac
     3961 ctgtatttcc agggagcagc ctcgatggat aagaaatact caataggctt agatatcggc
     4021 acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc
     4081 aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta
     4141 tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg tagaaggtat
     4201 acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga gatggcgaaa
     4261 gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag
     4321 catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat
     4381 ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc
     4441 ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga
     4501 gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac
     4561 aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt
     4621 tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag
     4681 aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt
     4741 aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat
     4801 gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca
     4861 gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata
     4921 actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg
     4981 actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt
     5041 gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt
     5101 tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa
     5161 ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat
     5221 caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt
     5281 ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt
     5341 ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca
     5401 attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt
     5461 gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc aaaacatagt
     5521 ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata tgttactgaa
     5581 ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt tgatttactc
     5641 ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt caaaaaaata
     5701 gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc ttcattaggt
     5761 acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa tgaagaaaat
     5821 gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag ggagatgatt
     5881 gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa acagcttaaa
     5941 cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg tattagggat
     6001 aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc caatcgcaat
     6061 tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca aaaagcacaa
     6121 gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg tagccctgct
     6181 attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa agtaatgggg
     6241 cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac aactcaaaag
     6301 ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga
     6361 agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga aaagctctat
     6421 ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga tattaatcgt
     6481 ttaagtgatt atgatgtcga tcacattgtt ccacaaagtt tccttaaaga cgattcaata
     6541 gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa cgttccaagt
     6601 gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc caagttaatc
     6661 actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat
     6721 aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa gcatgtggca
     6781 caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact tattcgagag
     6841 gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga tttccaattc
     6901 tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct aaatgccgtc
     6961 gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt ctatggtgat
     7021 tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat aggcaaagca
     7081 accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga aattacactt
     7141 gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac tggagaaatt
     7201 gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat gccccaagtc
     7261 aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc aattttacca
     7321 aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa aaaatatggt
     7381 ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg
     7441 aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat ggaaagaagt
     7501 tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga agttaaaaaa
     7561 gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg
     7621 atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc aagcaaatat
     7681 gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc agaagataac
     7741 gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat tattgagcaa
     7801 atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa agttcttagt
     7861 gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat tattcattta
     7921 tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac aacaattgat
     7981 cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca tcaatccatc
     8041 actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacgg gtcacctaag
     8101 aaaaaacgaa aagttgagga tcctaaaaag aaacgaaaag ttgatagcgg ttcagagacc
     8161 ccaggaacta gcgagagcgc tacaccggaa tcggcggaag cgggtatcac cggcacgtgg
     8221 tacaaccagt ctggttctac cttcaccgtt accgcgggtg cggacggtaa cctgaccggt
     8281 cagtacgaaa accgtgcgca gggcactggt tgccagaact ctccgtacac cctgaccggt
     8341 cgttacaacg gtaccaaact ggaatggcgt gttgaatgga acaactctac cgaaaactgc
     8401 cactctcgta ccgaatggcg tggtcagtac cagggtggtg cggaagcgcg tatcaacacc
     8461 cagtggaacc tgacctacga aggtggttct ggtccggcga ccgaacaggg tcaggacacc
     8521 ttcaccaaag ttaaaccgtc tgcggcgtct taagcggccg cactcgagca ccaccaccac
     8581 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     8641 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     8701 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     8761 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     8821 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     8881 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     8941 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     9001 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     9061 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     9121 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     9181 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     9241 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     9301 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     9361 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     9421 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     9481 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     9541 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     9601 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     9661 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     9721 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     9781 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     9841 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     9901 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     9961 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
    10021 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
    10081 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
    10141 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
    10201 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
    10261 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
    10321 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
    10381 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
    10441 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
    10501 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
    10561 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
    10621 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
    10681 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
    10741 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
    10801 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
    10861 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
    10921 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
    10981 gaagcggaag
//
