LOCUS       sequence-197576-       10010 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cas9 from S.pyogenes with CMV-mcherry cassette, and cloning backbone
            for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10010)
  AUTHORS   Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J
  TITLE     Correction of a genetic disease in mouse via use of CRISPR-Cas9.
  JOURNAL   Cell Stem Cell. 2013 Dec 5;13(6):659-62. doi: 
            10.1016/j.stem.2013.10.016.
  PUBMED    24315440
REFERENCE   2  (bases 1 to 10010)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stem.2013.10"; journalName: "Cell Stem Cell"; date: 
            "2013-12-5- 5"; volume: "13"; issue: "6"; pages: "659-62"
COMMENT     Sequence Label: px330-mcherry
FEATURES             Location/Qualifiers
     source          1..10010
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1013..1240
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1252..1261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1261..1326
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1333..1353
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1378..5478
                     /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             1378..5478
                     /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             5479..5526
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5554..5571)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5560..5767
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    5778..5859
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5803..5822
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    5983..6048
                     /label=MCS
                     /note="multiple cloning site"
     CDS             complement(6049..6756)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6324..6343)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6593..6611
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6753..6762
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6801..7004)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6830..6850)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7005..7308
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      7488..7943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7575..7594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7785..7806
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7960..7979)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8079..8101
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8139..8157)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8225..8329
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8330..8398,8399..9190)
                     /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(8548..8567)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9361..9949
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9850..9869
                     /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 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
      901 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
      961 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
     1021 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     1081 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     1141 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     1201 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg
     1261 gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac
     1321 gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac
     1381 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
     1441 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
     1501 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     1561 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     1621 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     1681 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     1741 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     1801 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     1861 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     1921 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     1981 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     2041 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     2101 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     2161 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     2221 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     2281 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     2341 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     2401 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     2461 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     2521 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     2581 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     2641 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     2701 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     2761 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     2821 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     2881 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     2941 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     3001 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     3061 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     3121 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     3181 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     3241 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     3301 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     3361 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     3421 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     3481 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     3541 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     3601 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     3661 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     3721 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     3781 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     3841 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     3901 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     3961 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     4021 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     4081 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     4141 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     4201 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     4261 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     4321 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     4381 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     4441 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     4501 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     4561 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     4621 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     4681 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     4741 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     4801 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     4861 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     4921 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     4981 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     5041 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     5101 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     5161 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     5221 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     5281 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     5341 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     5401 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     5461 tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
     5521 aaaaagtaag aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     5581 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5641 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5701 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     5761 ctggggagcg gccgcatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     5821 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     5881 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     5941 aaatgtggta tggctgatta tgatcagtta tctagatccg gtggatcccg ggcccgcggt
     6001 accgtcgact gcagaattcg aagcttgagc tcgagatctg agtccggact tgtacagctc
     6061 gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg tactgttcca cgatggtgta
     6121 gtcctcgttg tgggaggtga tgtccaactt gatgttgacg ttgtaggcgc cgggcagctg
     6181 cacgggcttc ttggccttgt aggtggtctt gacctcagcg tcgtagtggc cgccgtcctt
     6241 cagcttcagc ctctgcttga tctcgccctt cagggcgccg tcctcggggt acatccgctc
     6301 ggaggaggcc tcccagccca tggtcttctt ctgcattacg gggccgtcgg aggggaagtt
     6361 ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg agtcctgggt
     6421 cacggtcacc acgccgccgt cctcgaagtt catcacgcgc tcccatttga agccctcggg
     6481 gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg ccttggagcc
     6541 gtacatgaac tgaggggaca ggatgtccca ggcgaagggc agggggccac ccttggtcac
     6601 cttcagcttg gcggtctggg tgccctcgta ggggcggccc tcgccctcgc cctcgatctc
     6661 gaactcgtgg ccgttcacgg agccctccat gtgcaccttg aagcgcatga actccttgat
     6721 gatggccatg ttatcctcct cgcccttgct caccatggtg gcgaccggta gcgctagcgg
     6781 atctgacggt tcactaaacc agctctgctt atatagacct cccaccgtac acgcctaccg
     6841 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
     6901 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
     6961 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
     7021 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
     7081 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     7141 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     7201 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     7261 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     7321 tatgggctat gaactaatga ccccgtaatt gattactatg cggccgcaaa tgggcggcct
     7381 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     7441 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     7501 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     7561 cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     7621 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     7681 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     7741 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     7801 aactggaaca acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc
     7861 gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     7921 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     7981 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     8041 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     8101 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     8161 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     8221 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     8281 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     8341 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     8401 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     8461 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     8521 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     8581 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     8641 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     8701 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     8761 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     8821 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     8881 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     8941 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     9001 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
     9061 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     9121 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     9181 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     9241 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     9301 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     9361 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     9421 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     9481 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     9541 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     9601 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     9661 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     9721 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     9781 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     9841 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     9901 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     9961 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
