LOCUS       sequence-340296-        9131 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A knockout vector for the dog Hbegf..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9131)
  AUTHORS   Lin S, Hirayama D, Maryu G, Matsuda K, Hino N, Deguchi E, Aoki K, 
            Iwamoto R, Terai K, Matsuda M
  TITLE     Redundant roles of EGFR ligands in the ERK activation waves during 
            collective cell migration.
  JOURNAL   Life Sci Alliance. 2021 Oct 19;5(1). pii: 5/1/e202101206. doi: 
            10.26508/lsa.202101206. Print 2022 Jan.
  PUBMED    34667080
REFERENCE   2  (bases 1 to 9131)
  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: "Life Sci 
            Alliance."; date: "2021-10-19"; volume: "5(1). pii: 5/1/e202101206. 
            doi"; pages: " 10.26508/lsa.202101206. Print 2022 Jan"
COMMENT     Sequence Label: pX459-dHbegf
FEATURES             Location/Qualifiers
     source          1..9131
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          118..345
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      357..366
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             366..431
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             438..458
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             483..4583
                     /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             483..4583
                     /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             4584..4631
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4647..4700
                     /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             4701..5300
                     /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(4701..4720)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(5328..5345)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5334..5541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5550..5690
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5550..5679
                     /label=AAV2 ITR
     rep_origin      5765..6220
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5852..5871)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6062..6083
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6237..6256)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6356..6378
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6416..6434)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6502..6606
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6607..6675,6676..7467)
                     /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(6825..6844)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7638..8226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8127..8146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8288..8536
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8288..8308
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8459..8478
                     /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 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
       61 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      121 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      181 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      241 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
      301 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca
      361 ccatggacta taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg
      421 atgacgataa gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
      481 ccgacaagaa gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga
      541 tcaccgacga gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc
      601 acagcatcaa gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg
      661 ccacccggct gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct
      721 atctgcaaga gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac
      781 tggaagagtc cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca
      841 acatcgtgga cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga
      901 aactggtgga cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca
      961 tgatcaagtt ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg
     1021 tggacaagct gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca
     1081 tcaacgccag cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac
     1141 ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc
     1201 tgattgccct gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg
     1261 atgccaaact gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc
     1321 agatcggcga ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc
     1381 tgctgagcga catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta
     1441 tgatcaagag atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc
     1501 agcagctgcc tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg
     1561 gctacattga cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg
     1621 aaaagatgga cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga
     1681 agcagcggac cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg
     1741 ccattctgcg gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg
     1801 agaagatcct gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca
     1861 gattcgcctg gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag
     1921 tggtggacaa gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga
     1981 acctgcccaa cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt
     2041 ataacgagct gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga
     2101 gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg
     2161 tgaagcagct gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct
     2221 ccggcgtgga agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta
     2281 tcaaggacaa ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc
     2341 tgaccctgac actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc
     2401 acctgttcga cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca
     2461 ggctgagccg gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg
     2521 atttcctgaa gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca
     2581 gcctgacctt taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc
     2641 acgagcacat tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag
     2701 tgaaggtggt ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga
     2761 tcgaaatggc cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa
     2821 tgaagcggat cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg
     2881 tggaaaacac ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg
     2941 atatgtacgt ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata
     3001 tcgtgcctca gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg
     3061 acaagaaccg gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga
     3121 actactggcg gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga
     3181 ccaaggccga gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc
     3241 tggtggaaac ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca
     3301 ctaagtacga cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca
     3361 agctggtgtc cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact
     3421 accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt
     3481 accctaagct ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga
     3541 tgatcgccaa gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca
     3601 acatcatgaa ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc
     3661 ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg
     3721 ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc
     3781 agacaggcgg cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg
     3841 ccagaaagaa ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct
     3901 attctgtgct ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga
     3961 aagagctgct ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact
     4021 ttctggaagc caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt
     4081 actccctgtt cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc
     4141 agaagggaaa cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc
     4201 actatgagaa gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac
     4261 agcacaagca ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga
     4321 tcctggccga cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc
     4381 ccatcagaga gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc
     4441 ctgccgcctt caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag
     4501 aggtgctgga cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg
     4561 acctgtctca gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa
     4621 aaaagaaaaa ggaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
     4681 tcgaggagaa tcctggccca atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     4741 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     4801 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     4861 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     4921 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     4981 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     5041 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     5101 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     5161 ccggggtgcc cgccttcctg gagacctccg cgccccacaa cctccccttc tacgagcggc
     5221 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     5281 cccgcaagcc cggtgcctga gaattctaac tagagctcgc tgatcagcct cgactgtgcc
     5341 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5401 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5461 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5521 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     5581 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     5641 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     5701 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     5761 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5821 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     5881 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5941 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     6001 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6061 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     6121 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     6181 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     6241 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6301 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6361 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     6421 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     6481 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6541 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6601 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6661 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6721 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6781 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6841 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6901 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6961 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     7021 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     7081 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7141 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7201 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7261 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7321 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7381 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7441 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     7501 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     7561 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7621 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7681 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7741 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7801 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7861 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7921 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7981 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     8041 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     8101 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     8161 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     8221 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     8281 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     8341 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     8401 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     8461 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     8521 gaaaggacga aacaccgctc ccaccgaatc cacggacgtt 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 gcgggggggg g
//
