LOCUS       sequence-57166-e       10254 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual expression plasmid of human codon-optimized SpCas9 and a gRNA 
            to the human Emx1 locus, can be used to test SpCas9 cleavage in cell
            lines of choice..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10254)
  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 10254)
  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: 
            pX261-U6-DR-hEmx1-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro
FEATURES             Location/Qualifiers
     source          1..10254
                     /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   318..353
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     enhancer        404..707
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        709..986
                     /label=chicken beta-actin promoter
     intron          987..1215
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1227..1236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1236..1301
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1308..1328
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1353..5453
                     /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             1353..5453
                     /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             5454..5501
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5529..5546)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5535..5742
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5756..5970
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     5891..5910
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        5981..6059
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     primer_bind     6133..6154
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    6230..6263
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        6324..6820
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(6359..6382)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     6753..6772
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             6860..7459
                     /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(6860..6879)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     7356..7376
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     rep_origin      7732..8187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7819..7838)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8029..8050
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8204..8223)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8323..8345
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8383..8401)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8469..8573
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8574..8642,8643..9434)
                     /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(8792..8811)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9605..10193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10094..10113
                     /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 ccaaaacgga agggcctgag
      301 tccgagcaga agaagaagtt ttagagctat gctgttttga atggtcccaa aactttttct
      361 agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca taacttacgg
      421 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      481 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      541 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      601 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      661 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
      721 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
      781 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
      841 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      901 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      961 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc
     1021 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
     1081 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
     1141 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
     1201 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
     1261 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
     1321 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
     1381 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1441 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1501 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     1561 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     1621 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     1681 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     1741 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1801 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1861 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1921 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1981 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     2041 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     2101 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     2161 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     2221 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     2281 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     2341 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     2401 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2461 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     2521 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     2581 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     2641 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     2701 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     2761 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2821 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2881 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     2941 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     3001 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     3061 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     3121 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     3181 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     3241 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     3301 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     3361 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3421 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3481 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     3541 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     3601 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     3661 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     3721 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3781 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3841 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3901 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3961 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     4021 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     4081 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     4141 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     4201 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     4261 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     4321 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     4381 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     4441 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4501 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     4561 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     4621 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     4681 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     4741 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     4801 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     4861 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     4921 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     4981 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     5041 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     5101 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     5161 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     5221 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     5281 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     5341 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     5401 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacaagcgtc
     5461 ctgctgctac taagaaagct ggtcaagcta agaaaaagaa ataagaattc ctagagctcg
     5521 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     5581 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     5641 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     5701 caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc aattcgaacg
     5761 ctgacgtcat caacccgctc caaggaatcg cgggcccagt gtcactaggc gggaacaccc
     5821 agcgcgcgtg cgccctggca ggaagatggc tgtgagggac aggggagtgg cgccctgcaa
     5881 tatttgcatg tcgctatgtg ttctgggaaa tcaccataaa cgtgaaatgt ctttggattt
     5941 gggaatctta taagttctgt atgagaccac agatctagtt ggaaccattc aaaacagcat
     6001 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     6061 ttttggcgcc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga
     6121 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     6181 ggacgagctg tacaagtaag aattccgatc atattcaata acccttaata taacttcgta
     6241 taatgtatgc tatacgaagt tattaggtct gaagaggagt ttacgtccag ccaagcttag
     6301 gatctcgacc tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     6361 atgcgcttta gcagccccgc tggcacttgg cgctacacaa gtggcctctg gcctcgcaca
     6421 cattccacat ccaccggtag cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     6481 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     6541 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     6601 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     6661 gggctcagca gctgggaagg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg
     6721 ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc
     6781 tgccgcgctg ttctcctctt cctcatctcc gggcctttcg acctgcatcc atctagatct
     6841 cgagcagctg aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     6901 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     6961 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     7021 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     7081 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     7141 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     7201 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     7261 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     7321 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     7381 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     7441 ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc
     7501 acgaccccat gcatcgatga tatcagatcc ccgggatgca gaaattgatg atctattaaa
     7561 caataaagat gtccactaaa atggaagttt ttcctgtcat actttgttaa gaagggtgag
     7621 aacagagtac ctacattttg aatggaagga ttggagcggc gcctgatgcg gtattttctc
     7681 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     7741 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     7801 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     7861 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     7921 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     7981 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     8041 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     8101 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     8161 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     8221 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     8281 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     8341 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     8401 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     8461 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     8521 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     8581 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     8641 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     8701 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     8761 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     8821 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     8881 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     8941 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     9001 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     9061 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     9121 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     9181 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     9241 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     9301 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     9361 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     9421 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     9481 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     9541 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     9601 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     9661 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     9721 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     9781 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     9841 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     9901 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     9961 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
    10021 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
    10081 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
    10141 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
    10201 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
