LOCUS       sequence-171519-        8936 bp    DNA     linear   UNA 15-APR-2025
DEFINITION  Expresses sgRNA targeting human EGFR around codon T790.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8936)
  AUTHORS   Guernet A, Mungamuri SK, Cartier D, Sachidanandam R, Jayaprakash A, 
            Adriouch S, Vezain M, Charbonnier F, Rohkin G, Coutant S, Yao S, 
            Ainani H, Alexandre D, Tournier I, Boyer O, Aaronson SA, Anouar Y, 
            Grumolato L
  TITLE     CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and 
            Functional Analysis of Oncogenic Driver Mutations.
  JOURNAL   Mol Cell. 2016 Aug 4;63(3):526-38. doi: 
            10.1016/j.molcel.2016.06.017. Epub 2016 Jul 21.
  PUBMED    27453044
REFERENCE   2  (bases 1 to 8936)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2016.06.017"; journalName: "Mol Cell"; date: 
            "2016-08-4- 4"; volume: "63"; issue: "3"; pages: "526-38"
COMMENT     Sequence Label: sgEGFR(B)
FEATURES             Location/Qualifiers
     source          1..8936
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          8..235
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      247..256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             256..321
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             328..348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             373..4473
                     /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             373..4473
                     /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             4474..4521
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4537..4590
                     /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             4591..5190
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4591..4610)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(5218..5235)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5224..5431
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5440..5580
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5440..5569
                     /label=AAV2 ITR
     rep_origin      5655..6110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5742..5761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5952..5973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6127..6146)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6246..6268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6306..6324)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6392..6496
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6497..6565,6566..7357)
                     /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(6715..6734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7528..8116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8017..8036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8178..8426
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8178..8198
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8349..8368
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8448..8523
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8620..8905
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg
       61 ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc
      121 ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg
      181 tggggtatta atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg
      241 accggtgcca ccatggacta taaggaccac gacggagact acaaggatca tgatattgat
      301 tacaaagacg atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga
      361 gtcccagcag ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc
      421 tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac
      481 accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa
      541 acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac
      601 cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc
      661 ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc
      721 atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac
      781 ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc
      841 ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac
      901 aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag
      961 gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc
     1021 aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg
     1081 ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac
     1141 ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac
     1201 ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc
     1261 gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg
     1321 agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct
     1381 ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac
     1441 ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag
     1501 cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac
     1561 ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga
     1621 gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg
     1681 gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg
     1741 ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac
     1801 ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac
     1861 ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac
     1921 ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc
     1981 gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg
     2041 aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc
     2101 gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg
     2161 ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa
     2221 gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa
     2281 acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc
     2341 ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag
     2401 acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc
     2461 cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc
     2521 gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc
     2581 ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag
     2641 aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc
     2701 cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa
     2761 gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag
     2821 aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat
     2881 gtggaccata tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg
     2941 accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag
     3001 aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc
     3061 gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc
     3121 aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc
     3181 cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc
     3241 ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag
     3301 atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg
     3361 atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac
     3421 gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc
     3481 ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc
     3541 cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc
     3601 cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag
     3661 accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat
     3721 aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc
     3781 accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg
     3841 aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat
     3901 cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag
     3961 ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc
     4021 ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac
     4081 ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg
     4141 tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc
     4201 aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac
     4261 cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat
     4321 ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc
     4381 agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag
     4441 acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc
     4501 ggccaggcaa aaaagaaaaa ggaattcggc agtggagagg gcagaggaag tctgctaaca
     4561 tgcggtgacg tcgaggagaa tcctggccca atgaccgagt acaagcccac ggtgcgcctc
     4621 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4681 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     4741 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4801 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4861 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     4921 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     4981 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     5041 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccacaa cctccccttc
     5101 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5161 tggtgcatga cccgcaagcc cggtgcctga gaattctaac tagagctcgc tgatcagcct
     5221 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     5281 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5341 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5401 attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
     5461 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     5521 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     5581 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5641 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5701 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     5761 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5821 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5881 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5941 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     6001 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     6061 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     6121 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     6181 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6241 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     6301 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     6361 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6421 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6481 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6541 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6601 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6661 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6721 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6781 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6841 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6901 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6961 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     7021 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     7081 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     7141 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     7201 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7261 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7321 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7381 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7441 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7501 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7561 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7621 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7681 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7741 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7801 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7861 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7921 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7981 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     8041 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     8101 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     8161 gccttttgct cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     8221 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     8281 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     8341 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     8401 atatcttgtg gaaaggacga aacaccggag ctgcgtgatg agctgcagtt ttagagctag
     8461 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     8521 tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg
     8581 cgtgcgccaa ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa
     8641 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat
     8701 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     8761 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     8821 cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     8881 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactc
//
