LOCUS       sequence-242290-        9674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression plasmid for 3xNLS SpCas9 in pET-21a backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9674)
  AUTHORS   Wu Y, Zeng J, Roscoe BP, Liu P, Yao Q, Lazzarotto CR, Clement K, 
            Cole MA, Luk K, Baricordi C, Shen AH, Ren C, Esrick EB, Manis JP, 
            Dorfman DM, Williams DA, Biffi A, Brugnara C, Biasco L, Brendel C, 
            Pinello L, Tsai SQ, Wolfe SA, Bauer DE
  TITLE     Highly efficient therapeutic gene editing of human hematopoietic 
            stem cells.
  JOURNAL   Nat Med. 2019 May;25(5):776-783. doi: 10.1038/s41591-019-0401-y. 
            Epub 2019 Mar 25.
  PUBMED    30911135
REFERENCE   2  (bases 1 to 9674)
  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"; doi: 
            "10.1038/s41591-019-0401-y"; journalName: "Nat Med"; date: 
            "2019-05"; volume: "25"; issue: "5"; pages: "776-783"
COMMENT     Sequence Label: pET-21a_3xNLS_SpCas9_protein_expression
FEATURES             Location/Qualifiers
     source          1..9674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     964..983
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1003..1080)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1286..1305
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1389..1408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1389..1407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1408..1432
                     /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             1447..1469
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1477..1509
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1519..1545
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     CDS             1558..5661
                     /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             1558..5661
                     /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             5707..5727
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5740..5787
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5794..5811
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5864..5882)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5878..5925
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5962..6417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6049..6068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6259..6280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6443..6547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6548..6616,6617..7408)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6766..6785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7579..8167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8068..8087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8321..8338
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8353..8492
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8418..8437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8578..8600
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8594..8785)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(9594..9674,1..1002))
                     /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"
ORIGIN
        1 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
       61 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      121 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      181 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      241 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      301 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      361 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      421 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      481 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      541 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      601 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      661 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      721 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      781 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      841 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
      901 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
      961 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1021 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     1081 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     1141 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     1201 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     1261 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     1321 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     1381 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     1441 aataattttg tttaacttta agaaggagat atacatatgg ctagcatgac tggtggacag
     1501 caaatgggtc gcgccgctcc ggcagctaag aaaaagaaac tggatggcag cgtcgacatg
     1561 gataagaaat actcaatagg cttagatatc ggcacaaata gcgtcggatg ggcggtgatc
     1621 actgatgaat ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac
     1681 agtatcaaaa aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg
     1741 actcgtctca aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat
     1801 ctacaggaga ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt
     1861 gaagagtctt ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat
     1921 atagtagatg aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa
     1981 ttggtagatt ctactgataa agcggatttg cgcttaatct atttggcctt agcgcatatg
     2041 attaagtttc gtggtcattt tttgattgag ggagatttaa atcctgataa tagtgatgtg
     2101 gacaaactat ttatccagtt ggtacaaacc tacaatcaat tatttgaaga aaaccctatt
     2161 aacgcaagtg gagtagatgc taaagcgatt ctttctgcac gattgagtaa atcaagacga
     2221 ttagaaaatc tcattgctca gctccccggt gagaagaaaa atggcttatt tgggaatctc
     2281 attgctttgt cattgggttt gacccctaat tttaaatcaa attttgattt ggcagaagat
     2341 gctaaattac agctttcaaa agatacttac gatgatgatt tagataattt attggcgcaa
     2401 attggagatc aatatgctga tttgtttttg gcagctaaga atttatcaga tgctatttta
     2461 ctttcagata tcctaagagt aaatactgaa ataactaagg ctcccctatc agcttcaatg
     2521 attaaacgct acgatgaaca tcatcaagac ttgactcttt taaaagcttt agttcgacaa
     2581 caacttccag aaaagtataa agaaatcttt tttgatcaat caaaaaacgg atatgcaggt
     2641 tatattgatg ggggagctag ccaagaagaa ttttataaat ttatcaaacc aattttagaa
     2701 aaaatggatg gtactgagga attattggtg aaactaaatc gtgaagattt gctgcgcaag
     2761 caacggacct ttgacaacgg ctctattccc catcaaattc acttgggtga gctgcatgct
     2821 attttgagaa gacaagaaga cttttatcca tttttaaaag acaatcgtga gaagattgaa
     2881 aaaatcttga cttttcgaat tccttattat gttggtccat tggcgcgtgg caatagtcgt
     2941 tttgcatgga tgactcggaa gtctgaagaa acaattaccc catggaattt tgaagaagtt
     3001 gtcgataaag gtgcttcagc tcaatcattt attgaacgca tgacaaactt tgataaaaat
     3061 cttccaaatg aaaaagtact accaaaacat agtttgcttt atgagtattt tacggtttat
     3121 aacgaattga caaaggtcaa atatgttact gaaggaatgc gaaaaccagc atttctttca
     3181 ggtgaacaga agaaagccat tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt
     3241 aagcaattaa aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca
     3301 ggagttgaag atagatttaa tgcttcatta ggtacctacc atgatttgct aaaaattatt
     3361 aaagataaag attttttgga taatgaagaa aatgaagata tcttagagga tattgtttta
     3421 acattgacct tatttgaaga tagggagatg attgaggaaa gacttaaaac atatgctcac
     3481 ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc gttatactgg ttggggacgt
     3541 ttgtctcgaa aattgattaa tggtattagg gataagcaat ctggcaaaac aatattagat
     3601 tttttgaaat cagatggttt tgccaatcgc aattttatgc agctgatcca tgatgatagt
     3661 ttgacattta aagaagacat tcaaaaagca caagtgtctg gacaaggcga tagtttacat
     3721 gaacatattg caaatttagc tggtagccct gctattaaaa aaggtatttt acagactgta
     3781 aaagttgttg atgaattggt caaagtaatg gggcggcata agccagaaaa tatcgttatt
     3841 gaaatggcac gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg
     3901 aaacgaatcg aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt
     3961 gaaaatactc aattgcaaaa tgaaaagctc tatctctatt atctccaaaa tggaagagac
     4021 atgtatgtgg accaagaatt agatattaat cgtttaagtg attatgatgt cgatcacatt
     4081 gttccacaaa gtttccttaa agacgattca atagacaata aggtcttaac gcgttctgat
     4141 aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac
     4201 tattggagac aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg
     4261 aaagctgaac gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg
     4321 gttgaaactc gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact
     4381 aaatacgatg aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa
     4441 ttagtttctg acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac
     4501 catcatgccc atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat
     4561 ccaaaacttg aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg
     4621 attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat
     4681 atcatgaact tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct
     4741 ctaatcgaaa ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc
     4801 acagtgcgca aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag
     4861 acaggcggat tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct
     4921 cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat
     4981 tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa
     5041 gagttactag ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt
     5101 ttagaagcta aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat
     5161 agtctttttg agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa
     5221 aaaggaaatg agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat
     5281 tatgaaaagt tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag
     5341 cataagcatt atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt
     5401 ttagcagatg ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca
     5461 atacgtgaac aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc
     5521 gctgctttta aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa
     5581 gttttagatg ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat
     5641 ttgagtcagc taggaggtga caccggtggt ggtcccgggg gtggtgcggc cgcaggcagc
     5701 ggaagcccta agaaaaaacg aaaagttggc agcggaagca aaaggccggc ggccacgaaa
     5761 aaggccggcc aggcaaaaaa gaaaaagctc gagcaccacc accaccacca ctgagatccg
     5821 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     5881 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     5941 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     6001 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     6061 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     6121 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     6181 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     6241 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     6301 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     6361 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     6421 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6481 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6541 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6601 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6661 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     6721 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6781 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6841 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6901 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6961 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     7021 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     7081 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     7141 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7201 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7261 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     7321 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7381 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7441 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7501 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7561 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7621 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     7681 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     7741 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7801 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7861 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7921 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7981 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8041 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8101 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8161 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     8221 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     8281 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     8341 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     8401 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     8461 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     8521 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     8581 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     8641 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     8701 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     8761 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     8821 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     8881 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     8941 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     9001 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     9061 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     9121 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     9181 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     9241 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     9301 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     9361 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     9421 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     9481 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     9541 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     9601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     9661 ggagaggcgg tttg
//
