LOCUS       sequence-346238-        9105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To knockout APP gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9105)
  AUTHORS   Park H, Hundley FV, Yu Q, Overmyer KA, Brademan DR, Serrano L, Paulo
            JA, Paoli JC, Swarup S, Coon JJ, Gygi SP, Wade Harper J
  TITLE     Spatial snapshots of amyloid precursor protein intramembrane 
            processing via early endosome proteomics.
  JOURNAL   Nat Commun. 2022 Oct 16;13(1):6112. doi: 10.1038/s41467-022-33881-x.
  PUBMED    36245040
REFERENCE   2  (bases 1 to 9105)
  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: "Nat 
            Commun."; date: "2022-10-16"; volume: "13(1):6112. doi"; pages: " 
            10.1038/s41467-022-33881-x"
COMMENT     Sequence Label: PX459-APP-KO
FEATURES             Location/Qualifiers
     source          1..9105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          113..340
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      352..361
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             361..426
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             433..453
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             478..4578
                     /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             478..4578
                     /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             4579..4626
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4642..4695
                     /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             4696..5295
                     /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(4696..4715)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5192..5212
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5323..5340)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5329..5536
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5545..5685
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5545..5674
                     /label=AAV2 ITR
     rep_origin      5760..6215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5847..5866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6057..6078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6232..6251)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6351..6373
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6411..6429)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6497..6601
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6602..6670,6671..7462)
                     /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(6820..6839)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7633..8221
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8122..8141
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8283..8531
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8283..8303
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8454..8473
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8553..8628
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8725..9010
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
       61 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
      121 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
      181 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
      241 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
      301 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg
      361 gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac
      421 gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac
      481 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
      541 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
      601 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
      661 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
      721 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
      781 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
      841 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
      901 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
      961 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     1021 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     1081 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     1141 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     1201 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     1261 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     1321 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     1381 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     1441 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     1501 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     1561 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     1621 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     1681 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     1741 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     1801 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     1861 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     1921 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     1981 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     2041 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     2101 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     2161 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     2221 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     2281 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     2341 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     2401 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     2461 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     2521 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     2581 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     2641 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     2701 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     2761 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     2821 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     2881 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     2941 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     3001 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     3061 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     3121 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     3181 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     3241 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     3301 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     3361 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     3421 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     3481 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     3541 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     3601 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     3661 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     3721 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     3781 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     3841 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     3901 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     3961 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     4021 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     4081 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     4141 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     4201 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     4261 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     4321 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     4381 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     4441 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     4501 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     4561 tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
     4621 aaaaaggaat tcggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag
     4681 gagaatcctg gcccaatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4741 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4801 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4861 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4921 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4981 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     5041 caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc cgaccaccag
     5101 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     5161 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     5221 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     5281 aagcccggtg cctgagaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta
     5341 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     5401 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     5461 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata
     5521 gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     5581 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     5641 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     5701 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     5761 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5821 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5881 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5941 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     6001 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     6061 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     6121 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6181 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     6241 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     6301 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     6361 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     6421 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     6481 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6541 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6601 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6661 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6721 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6781 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6841 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6901 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6961 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7021 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     7081 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     7141 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7201 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7261 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     7321 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7381 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7441 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7501 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7561 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7621 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7681 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7741 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7801 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7861 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7921 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7981 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     8041 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     8101 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     8161 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     8221 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     8281 gtgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     8341 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     8401 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     8461 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     8521 gacgaaacac cggtcaacgg catcaggggt acgttttaga gctagaaata gcaagttaaa
     8581 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta
     8641 gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc gccaattctg
     8701 cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc cgcctggctg
     8761 accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga ctttccattg
     8821 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     8881 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc
     8941 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     9001 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     9061 cccaccccca attttgtatt tatttatttt ttaattattt tgtgc
//
