LOCUS       sequence-175005-        9121 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  CRISPR KO of Trp53.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 9121)
  AUTHORS   Wang Q, Zou Y, Nowotschin S, Kim SY, Li QV, Soh CL, Su J, Zhang C, 
            Shu W, Xi Q, Huangfu D, Hadjantonakis AK, Massague J
  TITLE     The p53 Family Coordinates Wnt and Nodal Inputs in Mesendodermal 
            Differentiation of Embryonic Stem Cells.
  JOURNAL   Cell Stem Cell. 2017 Jan 5;20(1):70-86. doi: 
            10.1016/j.stem.2016.10.002. Epub 2016 Nov 23.
  PUBMED    27889317
REFERENCE   2  (bases 1 to 9121)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.stem.2016.10.002"; journalName: "Cell Stem Cell"; date: 
            "2017-01-5- 5"; volume: "20"; issue: "1"; pages: "70-86"
COMMENT     Sequence Label: Px461-Cas9n-Trp53-sgRNA-alpha
FEATURES             Location/Qualifiers
     source          1..9121
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          5..232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      244..253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             253..318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             325..345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(370..393,394..396,397..4470)
                     /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             370..4470
                     /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             4471..4518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4534..4587
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4588..4590,4591..5301)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4588..4590,4591..5301)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4891..4910)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5238..5259
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5329..5346)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5335..5542
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5551..5691
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5551..5680
                     /label=AAV2 ITR
     rep_origin      5766..6221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5853..5872)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6063..6084
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6238..6257)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6357..6379
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6417..6435)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6503..6607
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6608..6676,6677..7468)
                     /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(6826..6845)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7639..8227
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8128..8147
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8289..8537
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8289..8309
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8460..8479
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8558..8633
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8730..9015
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
       61 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      121 tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg
      181 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc
      241 ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
      301 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
      361 ccagcagccg acaagaagta cagcatcggc ctggccatcg gcaccaactc tgtgggctgg
      421 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
      481 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
      541 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
      601 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
      661 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
      721 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
      781 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
      841 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
      901 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
      961 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     1021 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     1081 ggcaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     1141 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     1201 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     1261 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     1321 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     1381 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     1441 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     1501 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     1561 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     1621 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     1681 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     1741 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     1801 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     1861 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     1921 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     1981 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     2041 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     2101 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     2161 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     2221 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     2281 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     2341 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     2401 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     2461 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     2521 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     2581 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     2641 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     2701 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     2761 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     2821 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     2881 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     2941 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     3001 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     3061 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     3121 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     3181 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     3241 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     3301 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     3361 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     3421 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     3481 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     3541 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     3601 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     3661 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     3721 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     3781 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     3841 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     3901 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     3961 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4021 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     4081 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     4141 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     4201 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     4261 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     4321 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     4381 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     4441 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     4501 caggcaaaaa agaaaaagga attcggcagt ggagagggca gaggaagtct gctaacatgc
     4561 ggtgacgtcg aggagaatcc tggcccagtg agcaagggcg aggagctgtt caccggggtg
     4621 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     4681 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     4741 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     4801 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     4861 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     4921 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4981 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     5041 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     5101 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     5161 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     5221 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     5281 ggcatggacg agctgtacaa ggaattctaa ctagagctcg ctgatcagcc tcgactgtgc
     5341 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     5401 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     5461 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     5521 agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag ttggccactc
     5581 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     5641 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     5701 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     5761 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     5821 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct
     5881 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     5941 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag
     6001 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     6061 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga
     6121 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa
     6181 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac
     6241 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     6301 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     6361 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct
     6421 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     6481 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     6541 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     6601 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     6661 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     6721 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     6781 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6841 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6901 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6961 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     7021 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     7081 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     7141 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     7201 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7261 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7321 tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     7381 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     7441 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     7501 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     7561 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     7621 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     7681 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     7741 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     7801 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7861 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7921 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7981 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     8041 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     8101 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     8161 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     8221 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     8281 tcacatgtga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     8341 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     8401 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     8461 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     8521 ggaaaggacg aaacaccgac cctgtcaccg agaccccgtt ttagagctag aaatagcaag
     8581 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     8641 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa
     8701 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct
     8761 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
     8821 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     8881 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     8941 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     9001 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     9061 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     9121 g
//
