LOCUS       sequence-313936-       12814 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12814)
  AUTHORS   Wang B, Wang Z, Wang D, Zhang B, Ong SG, Li M, Yu W, Wang Y
  TITLE     krCRISPR: an easy and efficient strategy for generating conditional 
            knockout of essential genes in cells.
  JOURNAL   J Biol Eng. 2019 Apr 24;13:35. doi: 10.1186/s13036-019-0150-y. 
            eCollection 2019.
  PUBMED    31049076
REFERENCE   2  (bases 1 to 12814)
  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: "J Biol 
            Eng."; date: "2019-04-24"; volume: "13:35. doi"; pages: " 
            10.1186/s13036-019-0150-y. eCollection 2019"
COMMENT     Sequence Label: KO plasmid
FEATURES             Location/Qualifiers
     source          1..12814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    393..510
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        579..790
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      810..819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             819..842
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             849..869
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             894..4994
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             894..4994
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4995..5042
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5058..5114
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(5115..5735,5736..6122)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(5188..5207)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     polyA_signal    6153..6287
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6177..6196)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6231..6250
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(6430..6452)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(10700..10718)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        10786..10890
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10891..10959,10960..11751)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(11109..11128)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11922..12510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12411..12430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(12699..12719)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg agaagagcga tgctcttcgg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgaattctag
      361 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
      421 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
      481 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
      541 ctttggcgcc ggctcgagtg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
      601 agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg
      661 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
      721 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
      781 cagaacacag gtgtcgtgac gcgggatccg ccaccatgga ttacaaagac gatgacgata
      841 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
      901 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
      961 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1021 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1081 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1141 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1201 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1261 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1321 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1381 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1441 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1501 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     1561 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     1621 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     1681 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     1741 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     1801 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     1861 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     1921 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     1981 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2041 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2101 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2161 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2221 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2281 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2341 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2401 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2461 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     2521 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     2581 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     2641 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     2701 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     2761 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     2821 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     2881 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     2941 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3001 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3061 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3121 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3181 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3241 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3301 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3361 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3421 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3481 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     3541 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     3601 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     3661 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     3721 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     3781 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     3841 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     3901 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     3961 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4021 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4081 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4141 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4201 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4261 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4321 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4381 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4441 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4501 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     4561 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     4621 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     4681 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     4741 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     4801 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     4861 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     4921 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     4981 agctgggagg cgacaagcgt cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga
     5041 aagctagcgg cagcggcgcc accaacttca gcctgctgaa gcaggccggc gacgtggagg
     5101 agaaccccgg ccccatgtct agattagata aaagtaaagt gattaacagc gcattagagc
     5161 tgcttaatga ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg
     5221 tagagcagcc tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag
     5281 ccattgagat gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc
     5341 aagatttttt acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg
     5401 gagcaaaagt acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc
     5461 aattagcctt tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg
     5521 ctgtggggca ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag
     5581 aagaaaggga aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat
     5641 tatttgatca ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg
     5701 gattagaaaa acaacttaaa tgtgaaagtg ggtccgcgta cagccgcgcg cgtacgaaaa
     5761 acaattacgg gtctaccatc gagggcctgc tcgatctccc ggacgacgac gcccccgaag
     5821 aggcggggct ggcggctccg cgcctgtcct ttctccccgc gggacacacg cgcagactgt
     5881 cgacggcccc cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg
     5941 tggcgatggc gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg
     6001 attccccggg tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgg
     6061 ccgacttcga gtttgagcag atgtttaccg atgcccttgg aattgacgag tacggtgggt
     6121 agcttaagaa ccgctcgagg ccggcaaggc cggatccaga catgataaga tacattgatg
     6181 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     6241 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     6301 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     6361 acctctacaa atgtggtatg gctgattatg atccggctgc ctcgcgcgtt tcggtgatga
     6421 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     6481 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     6541 agccatgagg tcgactctag aggatcgatc cccgccccgg acgaactaaa cctgactacg
     6601 acatctctgc cccttcttcg cggggcagtg catgtaatcc cttcagttgg ttggtacaac
     6661 ttgccaactg ggccctgttc cacatgtgac acgggggggg accaaacaca aaggggttct
     6721 ctgactgtag ttgacatcct tataaatgga tgtgcacatt tgccaacact gagtggcttt
     6781 catcctggag cagactttgc agtctgtgga ctgcaacaca acattgcctt tatgtgtaac
     6841 tcttggctga agctcttaca ccaatgctgg gggacatgta cctcccaggg gcccaggaag
     6901 actacgggag gctacaccaa cgtcaatcag aggggcctgt gtagctaccg ataagcggac
     6961 cctcaagagg gcattagcaa tagtgtttat aaggccccct tgttaaccct aaacgggtag
     7021 catatgcttc ccgggtagta gtatatacta tccagactaa ccctaattca atagcatatg
     7081 ttacccaacg ggaagcatat gctatcgaat tagggttagt aaaagggtcc taaggaacag
     7141 cgatatctcc caccccatga gctgtcacgg ttttatttac atggggtcag gattccacga
     7201 gggtagtgaa ccattttagt cacaagggca gtggctgaag atcaaggagc gggcagtgaa
     7261 ctctcctgaa tcttcgcctg cttcttcatt ctccttcgtt tagctaatag aataactgct
     7321 gagttgtgaa cagtaaggtg tatgtgaggt gctcgaaaac aaggtttcag gtgacgcccc
     7381 cagaataaaa tttggacggg gggttcagtg gtggcattgt gctatgacac caatataacc
     7441 ctcacaaacc ccttgggcaa taaatactag tgtaggaatg aaacattctg aatatcttta
     7501 acaatagaaa tccatggggt ggggacaagc cgtaaagact ggatgtccat ctcacacgaa
     7561 tttatggcta tgggcaacac ataatcctag tgcaatatga tactggggtt attaagatgt
     7621 gtcccaggca gggaccaaga caggtgaacc atgttgttac actctatttg taacaagggg
     7681 aaagagagtg gacgccgaca gcagcggact ccactggttg tctctaacac ccccgaaaat
     7741 taaacggggc tccacgccaa tggggcccat aaacaaagac aagtggccac tctttttttt
     7801 gaaattgtgg agtgggggca cgcgtcagcc cccacacgcc gccctgcggt tttggactgt
     7861 aaaataaggg tgtaataact tggctgattg taaccccgct aaccactgcg gtcaaaccac
     7921 ttgcccacaa aaccactaat ggcaccccgg ggaatacctg cataagtagg tgggcgggcc
     7981 aagatagggg cgcgattgct gcgatctgga ggacaaatta cacacacttg cgcctgagcg
     8041 ccaagcacag ggttgttggt cctcatattc acgaggtcgc tgagagcacg gtgggctaat
     8101 gttgccatgg gtagcatata ctacccaaat atctggatag catatgctat cctaatctat
     8161 atctgggtag cataggctat cctaatctat atctgggtag catatgctat cctaatctat
     8221 atctgggtag tatatgctat cctaatttat atctgggtag cataggctat cctaatctat
     8281 atctgggnta gcatatgcta tcctanatct rkrtmtgsrt akkmtatsct awyctatatc
     8341 tgkrtangca taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat
     8401 ctgggtagta tatgctatcc taatttatat ctgggtagca taggctatcc taatctatat
     8461 ctgggtagca tatgctatcc taatctatat ctgggtagta tatgctatcc taatctgtat
     8521 ccgggtagca tatgctatcc tcatgcatat acagtcagca tatgataccc agtagtagag
     8581 tgggagtgct atcctttgca tatgccgcca cctcccaagg gggcgtgaat tttcgctgct
     8641 tgtccttttc ctgctggttg ctcccattct taggtgaatt taaggaggcc aggctaaagc
     8701 cgtcgcatgt ctgattgctc accaggtaaa tgtcgctaat gttttccaac gcgagaaggt
     8761 gttgagcgcg gagctgagtg acgtgacaac atgggtatgc ccaattgccc catgttggga
     8821 ggacgaaaat ggtgacaaga cagatggcca gaaatacacc aacagcacgc atgatgtcta
     8881 ctggggattt attctttagt gcgggggaat acacggcttt taatacgatt gagggcgtct
     8941 cctaacaagt tacatcactc ctgcccttcc tcaccctcat ctccatcacc tccttcatct
     9001 ccgtcatctc cgtcatcacc ctccgcggca gccccttcca ccataggtgg aaaccaggga
     9061 ggcaaatcta ctccatcgtc aaagctgcac acagtcaccc tgatattgca ggtaggagcg
     9121 ggctttgtca taacaaggtc cttaatcgca tccttcaaaa cctcagcaaa tatatgagtt
     9181 tgtaaaaaga ccatgaaata acagacaatg gactccctta gcgggccagg ttgtgggccg
     9241 ggtccagggg ccattccaaa ggggagacga ctcaatggtg taagacgaca ttgtggaata
     9301 gcaagggcag ttcctcgcct taggttgtaa agggaggtct tactacctcc atatacgaac
     9361 acaccggcga cccaagttcc ttcgtcggta gtcctttcta cgtgactcct agccaggaga
     9421 gctcttaaac cttctgcaat gttctcaaat ttcgggttgg aacctccttg accacgatgc
     9481 tttccaaacc accctccttt tttgcgcctg cctccatcac cctgaccccg gggtccagtg
     9541 cttgggcctt ctcctgggtc atctgcgggg ccctgctcta tcgctcccgg gggcacgtca
     9601 ggctcaccat ctgggccacc ttcttggtgg tattcaaaat aatcggcttc ccctacaggg
     9661 tggaaaaatg gccttctacc tggagggggc ctgcgcggtg gagacccgga tgatgatgac
     9721 tgactactgg gactcctggg cctcttttct ccacgtccac gacctctccc cctggctctt
     9781 tcacgacttc cccccctggc tctttcacgt cctctacccc ggcggcctcc actacctcct
     9841 cgaccccggc ctccactacc tcctcgaccc cggcctccac tgcctcctcg accccggcct
     9901 ccacctcctg ctcctgcccc tcctgctcct gcccctccyc ctsctcctgc nnntcctgcc
     9961 cctcctgctc ctgcccctcc cgctcctgct cctgctcctg ttccaccgtg ggtccctttg
    10021 cagccaatgc aacttggacg tttttggggt ctccggacac catctctatg tcttggccct
    10081 gatcctgagc cgcccggggc tcctggtctt ccgcctcctc gtcctcgtcc tcttccccgt
    10141 cctcgtccat ggttatcacc ccctcttctt tgaggtccac tgccgccgga gccttctggt
    10201 ccagatgtgt ctcccttctc tcctaggcca tttccaggtc ctgtacctgg cccctcgtca
    10261 gacatgattc acactaaaag agatcaatag acatctttat tagacgacgc tcagtgaata
    10321 cagggagtgc agactcctgc cccctccaac agccccccca ccctcatccc cttcatggtc
    10381 gctgtcagac agatccaggt ctgaaaattc cccatcctcc gaaccatcct cgtcctcatc
    10441 accaattact cgcagcccgg aaaactcccg ctgaacatcc tcaagatttg cgtcctgagc
    10501 ctcaagccag gcctcaaatt cctcgtcccc ctttttgctg gacggtaggg atggggattc
    10561 tcgggacccc tcctcttcct cttcaaggtc accagacaga gatgctactg gggcaacgga
    10621 agaaaagctg ggtgcggcct gtgaggatca gcttatcgat gataagctgt caaacatgag
    10681 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
    10741 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
    10801 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
    10861 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
    10921 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
    10981 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
    11041 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
    11101 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
    11161 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
    11221 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
    11281 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
    11341 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
    11401 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
    11461 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
    11521 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
    11581 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
    11641 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
    11701 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
    11761 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
    11821 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
    11881 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
    11941 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
    12001 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
    12061 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
    12121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
    12181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
    12241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
    12301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
    12361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
    12421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
    12481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
    12541 ctggcctttt gctggccttg aagctgtccc tgatggtcgt catctacctg cctggacagc
    12601 atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag
    12661 gccatccagc ctcgcgtcga actagatgat ccggctgtgg aatgtgtgtc agttagggtg
    12721 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatt atggtacctt
    12781 tcagacccac ctcccaaccc cgaggggacc caga
//
