LOCUS       sequence-59273-e       10151 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid separately encodes a human codon-optimized SpCas9 
            nickase, a tracrRNA and customizable crRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10151)
  AUTHORS   Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, 
            Jiang W, Marraffini LA, Zhang F
  TITLE     Multiplex Genome Engineering Using CRISPR/Cas Systems.
  JOURNAL   Science. 2013 Jan 3.
  PUBMED    23287718
REFERENCE   2  (bases 1 to 10151)
  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: "Science. 
            2013 Jan 3."
COMMENT     Sequence Label: 
            pX334-U6-DR-BB-DR-Cbh-NLS-hSpCas9n(D10A)-NLS-H1-shorttracr-PGK-puro
FEATURES             Location/Qualifiers
     source          1..10151
                     /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"
     repeat_region   252..287
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   304..339
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     enhancer        390..675
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        677..954
                     /label=chicken beta-actin promoter
     intron          955..1183
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             1204..1230
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1234..1254
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1264..1287,1288..1290,1291..5364)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1264..5364
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5368..5388
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5425..5442)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5431..5638
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5652..5866
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     5787..5806
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        5877..5955
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     6029..6050
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    6126..6159
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6220..6717
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6255..6278)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6650..6669
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6757..7356
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6757..6776)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7253..7273
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     rep_origin      7629..8084
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7716..7735)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7926..7947
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8101..8120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8220..8242
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8280..8298)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8366..8470
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8471..8539,8540..9331)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8689..8708)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9502..10090
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9991..10010
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggttttagag ctatgctgtt ttgaatggtc ccaaaacggg tcttcgagaa
      301 gacgttttag agctatgctg ttttgaatgg tcccaaaact ttttctagcg cgtgcgccaa
      361 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct
      421 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
      481 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      541 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      601 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      661 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
      721 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
      781 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
      841 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
      901 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
      961 gctgcgacgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     1021 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     1081 ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat
     1141 gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc
     1201 atgtacccat acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg
     1261 tccgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
     1321 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     1381 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     1441 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     1501 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     1561 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     1621 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     1681 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     1741 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     1801 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     1861 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     1921 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggcaac
     1981 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     2041 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     2101 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     2161 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     2221 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     2281 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     2341 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     2401 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     2461 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     2521 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     2581 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     2641 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     2701 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     2761 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     2821 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     2881 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     2941 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     3001 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     3061 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     3121 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     3181 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     3241 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     3301 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     3361 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     3421 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     3481 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     3541 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     3601 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     3661 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     3721 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     3781 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     3841 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     3901 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     3961 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     4021 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     4081 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     4141 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     4201 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     4261 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     4321 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     4381 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     4441 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     4501 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     4561 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     4621 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     4681 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     4741 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     4801 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     4861 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     4921 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     4981 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     5041 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     5101 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     5161 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     5221 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     5281 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     5341 gacctgtctc agctgggagg cgacagcccc aagaagaaga gaaaggtgga ggccagctaa
     5401 gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     5461 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     5521 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     5581 gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc
     5641 ggccgcaatt cgaacgctga cgtcatcaac ccgctccaag gaatcgcggg cccagtgtca
     5701 ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa gatggctgtg agggacaggg
     5761 gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct gggaaatcac cataaacgtg
     5821 aaatgtcttt ggatttggga atcttataag ttctgtatga gaccacagat ctagttggaa
     5881 ccattcaaaa cagcatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     5941 gcaccgagtc ggtgcttttt ggcgccgaca accactacct gagcacccag tccgccctga
     6001 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     6061 ggatcactct cggcatggac gagctgtaca agtaagaatt ccgatcatat tcaataaccc
     6121 ttaatataac ttcgtataat gtatgctata cgaagttatt aggtctgaag aggagtttac
     6181 gtccagccaa gcttaggatc tcgacctcga aattctaccg ggtaggggag gcgcttttcc
     6241 caaggcagtc tggagcatgc gctttagcag ccccgctggc acttggcgct acacaagtgg
     6301 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     6361 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     6421 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     6481 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     6541 ttgctccttc gctttctggg ctcagcagct gggaagggtg ggtccggggg cgggctcagg
     6601 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     6661 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     6721 tgcatccatc tagatctcga gcagctgaag cttaccatga ccgagtacaa gcccacggtg
     6781 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     6841 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     6901 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     6961 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     7021 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     7081 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     7141 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     7201 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     7261 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     7321 cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc
     7381 ccgaccgaaa ggagcgcacg accccatgca tcgatgatat cagatccccg ggatgcagaa
     7441 attgatgatc tattaaacaa taaagatgtc cactaaaatg gaagtttttc ctgtcatact
     7501 ttgttaagaa gggtgagaac agagtaccta cattttgaat ggaaggattg gagcggcgcc
     7561 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     7621 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     7681 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     7741 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     7801 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     7861 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     7921 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt
     7981 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     8041 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     8101 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     8161 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     8221 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     8281 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     8341 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     8401 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     8461 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     8521 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     8581 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     8641 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     8701 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     8761 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     8821 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     8881 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     8941 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     9001 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     9061 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     9121 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     9181 gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     9241 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     9301 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     9361 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     9421 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     9481 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     9541 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     9601 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     9661 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     9721 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     9781 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     9841 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     9901 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     9961 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
    10021 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
    10081 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
    10141 tgctcacatg t
//
