LOCUS       sequence-494457-        9211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR vector co-expressing Cas9 and a mouse Itga5 gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9211)
  AUTHORS   Heiden R, Hannig L, Kuo CJ, Ergun S, Braunger BM, Vallon M
  TITLE     WNT7A/B assemble a GPR124-RECK-LRP5/6 co-receptor complex to 
            activate beta-catenin signaling in brain endothelial cells.
  JOURNAL   J Biol Chem. 2025 Sep 4:110682. doi: 10.1016/j.jbc.2025.110682.
  PUBMED    40914247
REFERENCE   2  (bases 1 to 9211)
  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: "J Biol 
            Chem. 2025 Sep 4:110682. doi: 10.1016/j.jbc.2025.110682."
COMMENT     Sequence Label: pX459-Itga5 gRNA
FEATURES             Location/Qualifiers
     source          1..9211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(3098..3118)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(3125..3190)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      3190..3199
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          3211..3438
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     enhancer        3752..4037
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     misc_RNA        complement(4134..4209)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(4231..4479)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4289..4308)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4459..4479)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(4541..5129)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4621..4640)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5300..6091,6092..6160))
                     /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     5923..5942
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6161..6265)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6333..6351
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6389..6411)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6511..6530
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6547..7002)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6684..6705)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6896..6915
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   7077..7217
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7088..7217
                     /label=AAV2 ITR
     polyA_signal    7226..7433
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7422..7439
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(7467..8066)
                     /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(7550..7570)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     8047..8066
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(8067..8120)
                     /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             complement(8136..8183)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(8184..9211,1..3073))
                     /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             complement(join(8184..9211,1..3073))
                     /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"
ORIGIN
        1 cgctcttggc gatcatcttc cgcacgtcgt acaccttgta gtcgccgtac acgaactcgc
       61 tttccagctt agggtacttt ttgatcaggg cggttcccac gacggcgttc aggtaggcgt
      121 cgtgggcgtg gtggtagttg ttgatctcgc gcactttgta aaactggaaa tccttccgga
      181 aatcggacac cagcttggac ttcagggtga tcactttcac ttcccggatc agcttgtcat
      241 tctcgtcgta cttagtgttc atccgggagt ccaggatctg tgccacgtgc tttgtgatct
      301 gccgggtttc caccagctgt ctcttgatga agccggcctt atccagttcg ctcaggccgc
      361 ctctctcggc cttggtcaga ttgtcgaact ttctctgggt aatcagcttg gcgttcagca
      421 gctgccgcca gtagttcttc atcttcttca cgacctcttc ggagggcacg ttgtcgctct
      481 tgccccggtt cttgtcgctt ctggtcagca ccttgttgtc gatggagtcg tccttcagaa
      541 agctctgagg cacgatatgg tccacatcgt agtcggacag ccggttgatg tccagttcct
      601 ggtccacgta catatcccgc ccattctgca ggtagtacag gtacagcttc tcgttctgca
      661 gctgggtgtt ttccacgggg tgttctttca ggatctggct gcccagctct ttgatgccct
      721 cttcgatccg cttcattctc tcgcggctgt tcttctgtcc cttctgggtg gtctggttct
      781 ctctggccat ttcgatcacg atgttctcgg gcttgtgccg gcccatcact ttcacgagct
      841 cgtccaccac cttcactgtc tgcaggatgc ccttcttaat ggcggggctg ccggccagat
      901 tggcaatgtg ctcgtgcagg ctatcgccct ggccggacac ctgggctttc tggatgtcct
      961 ctttaaaggt caggctgtcg tcgtggatca gctgcatgaa gtttctgttg gcgaagccgt
     1021 cggacttcag gaaatccagg attgtcttgc cggactgctt gtcccggatg ccgttgatca
     1081 gcttccggct cagcctgccc cagccggtgt atctccgccg cttcagctgc ttcatcactt
     1141 tgtcgtcgaa caggtgggca taggttttca gccgttcctc gatcatctct ctgtcctcaa
     1201 acagtgtcag ggtcagcacg atatcttcca gaatgtcctc gttttcctca ttgtccagga
     1261 agtccttgtc cttgataatt ttcagcagat cgtggtatgt gcccagggag gcgttgaacc
     1321 gatcttccac gccggagatt tccacggagt cgaagcactc gattttcttg aagtagtcct
     1381 ctttcagctg cttcacggtc actttccggt tggtcttgaa cagcaggtcc acgatggcct
     1441 ttttctgctc gccgctcagg aaggcgggct ttctcattcc ctcggtcacg tatttcactt
     1501 tggtcagctc gttatacacg gtgaagtact cgtacagcag gctgtgcttg ggcagcacct
     1561 tctcgttggg caggttctta tcgaagttgg tcatccgctc gatgaagctc tgggcggaag
     1621 cgcccttgtc caccacttcc tcgaagttcc agggggtgat ggtttcctcg ctctttctgg
     1681 tcatccaggc gaatctgctg tttcccctgg ccagagggcc cacgtagtag gggatgcgga
     1741 aggtcaggat cttctcgatc ttttcccggt tgtccttcag gaatgggtaa aaatcttcct
     1801 gccgccgcag aatggcgtgc agctctccca ggtggatctg gtgggggatg ctgccgttgt
     1861 cgaaggtccg ctgcttccgc agcaggtcct ctctgttcag cttcacgagc agttcctcgg
     1921 tgccgtccat cttttccagg atgggcttga tgaacttgta gaactcttcc tggctggctc
     1981 cgccgtcaat gtagccggcg tagccgttct tgctctggtc gaagaaaatc tctttgtact
     2041 tctcaggcag ctgctgccgc acgagagctt tcagcagggt caggtcctgg tggtgctcgt
     2101 cgtatctctt gatcatagag gcgctcaggg gggccttggt gatctcggtg ttcactctca
     2161 ggatgtcgct cagcaggatg gcgtcggaca ggttcttggc ggccagaaac aggtcggcgt
     2221 actggtcgcc gatctgggcc agcaggttgt ccaggtcgtc gtcgtaggtg tccttgctca
     2281 gctgcagttt ggcatcctcg gccaggtcga agttgctctt gaagttgggg gtcaggccca
     2341 ggctcagggc aatcaggttt ccgaacaggc cattcttctt ctcgccgggc agctgggcga
     2401 tcagattttc cagccgtctg ctcttgctca gtctggcaga caggatggcc ttggcgtcca
     2461 cgccgctggc gttgatgggg ttttcctcga acagctggtt gtaggtctgc accagctgga
     2521 tgaacagctt gtccacgtcg ctgttgtcgg ggttcaggtc gccctcgatc aggaagtggc
     2581 cccggaactt gatcatgtgg gccagggcca gatagatcag ccgcaggtcg gccttgtcgg
     2641 tgctgtccac cagtttcttt ctcaggtggt agatggtggg gtacttctcg tggtaggcca
     2701 cctcgtccac gatgttgccg aagatggggt gccgctcgtg cttcttatcc tcttccacca
     2761 ggaaggactc ttccagtctg tggaagaagc tgtcgtccac cttggccatc tcgttgctga
     2821 agatctcttg cagatagcag atccggttct tccgtctggt gtatcttctt ctggcggttc
     2881 tcttcagccg ggtggcctcg gctgtttcgc cgctgtcgaa cagcagggct ccgatcaggt
     2941 tcttcttgat gctgtgccgg tcggtgttgc ccagcacctt gaatttcttg ctgggcacct
     3001 tgtactcgtc ggtgatcacg gcccagccca cagagttggt gccgatgtcc aggccgatgc
     3061 tgtacttctt gtcggctgct gggactccgt ggataccgac cttccgcttc ttctttgggg
     3121 ccatcttatc gtcatcgtct ttgtaatcaa tatcatgatc cttgtagtct ccgtcgtggt
     3181 ccttatagtc catggtggca ccggtccaac ctgaaaaaaa gtgatttcag gcaggtgctc
     3241 caggtaatta aacattaata ccccaccaac caaccatccc ttaaaccctt acctcttgct
     3301 cagctaatta cagcccggag gagaagggcc gtcccgcccg ctcacctgtg ggagtaacgc
     3361 ggtcagtcag agccggggcg ggcggcgcga ggcggcggcg gagcggggca cggggcgaag
     3421 gcagcgcgca gcgactcccg cccgccgcgc gcttcgcttt ttatagggcc gccgccgccg
     3481 ccgcctcgcc ataaaaggaa actttcggag cgcgccgctc tgattggctg ccgccgcacc
     3541 tctccgcctc cccccgcccc gccccccgcc cccccccccc cccccccccc cccccccccc
     3601 cccccccccc cccccccccc gggccccgcc cccccccccc ccccgccccc atcgctgcac
     3661 aaaataatta aaaaataaat aaatacaaaa ttgggggtgg ggaggggggg gagatgggga
     3721 gagtgaagca gaacgtgggg ctcacctcga ccatggtaat agcgatgact aatacgtaga
     3781 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcacaatgc caggcgggcc
     3841 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     3901 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     3961 gcgttactat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
     4021 ccgtaagtta tgtaacgggt acctctagag ccatttgtct gcagaattgg cgcacgcgct
     4081 aaaaacggac tagccttatt ttaacttgct atttctagct ctaaaacaaa aaagcaccga
     4141 ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg ctatttctag
     4201 ctctaaaaca cgattagatc tgtagaaagc ggtgtttcgt cctttccaca agatatataa
     4261 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca
     4321 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac
     4381 taatatcttt gtgtttacag tcaaattaat tccaattatc tctctaacag ccttgtatcg
     4441 tatatgcaaa tatgaaggaa tcatgggaaa taggccctca catgtgagca aaaggccagc
     4501 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     4561 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4621 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4681 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     4741 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4801 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4861 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4921 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4981 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5041 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5101 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5161 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5221 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5281 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5341 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5401 agggcttacc atctggcccc agtgctgcaa tgataccgcg gcttccacgc tcaccggctc
     5461 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     5521 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     5581 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     5641 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     5701 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     5761 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     5821 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     5881 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5941 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6001 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6061 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6121 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6181 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6241 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     6301 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     6361 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     6421 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     6481 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     6541 accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     6601 gctcattttt taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc
     6661 ccgagataga gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     6721 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     6781 cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     6841 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     6901 agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     6961 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg
     7021 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgcccctg
     7081 caggcagctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc
     7141 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
     7201 catcactagg ggttcctgcg gccgctcccc agcatgcctg ctattctctt cccaatcctc
     7261 ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg
     7321 cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa
     7381 ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc
     7441 tgatcagcga gctctagtta gaattctcag gcaccgggct tgcgggtcat gcaccaggtg
     7501 cgcggtcctt cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag
     7561 gggaggttgc ggggcgcgga ggtctccagg aaggcgggca ccccggcgcg ctcggccgcc
     7621 tccactccgg ggagcacgac ggcgctgccc agacccttgc cctggtggtc gggcgagact
     7681 ccgacggtgg ccaggaacca cgcgggctcc ttgggccggt gcggcgccag gaggccttcc
     7741 atctgttgct gcgcggccag ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg
     7801 gcgaacaccg cccccgcttc gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg
     7861 tcgtccgcga cccacacctt gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc
     7921 agctcggtga cccgctcgat gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag
     7981 tcggcgaacg cggcggcgag ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg
     8041 cgcaccgtgg gcttgtactc ggtcattggg ccaggattct cctcgacgtc accgcatgtt
     8101 agcagacttc ctctgccctc tccactgccg aattcctttt tcttttttgc ctggccggcc
     8161 tttttcgtgg ccgccggcct tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg
     8221 tacaggccgg tgatgctctg gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg
     8281 tacctcttcc ggtcgatggt ggtgtcaaag tacttgaagg cggcaggggc tcccagattg
     8341 gtcagggtaa acaggtggat gatattctcg gcctgctctc tgatgggctt atcccggtgc
     8401 ttgttgtagg cggacagcac tttgtccaga ttagcgtcgg ccaggatcac tctcttggag
     8461 aactcgctga tctgctcgat gatctcgtcc aggtagtgct tgtgctgttc cacaaacagc
     8521 tgtttctgct cattatcctc gggggagccc ttcagcttct catagtggct ggccaggtac
     8581 aggaagttca catatttgga gggcagggcc agttcgtttc ccttctgcag ttcgccggca
     8641 gaggccagca ttctcttccg gccgttttcc agctcgaaca gggagtactt aggcagcttg
     8701 atgatcaggt cctttttcac ttctttgtag cccttggctt ccagaaagtc gatgggattc
     8761 ttctcgaagc tgcttctttc catgatggtg atccccagca gctctttcac actcttcagt
     8821 ttcttggact tgcccttttc cactttggcc accaccagca cagaataggc cacggtgggg
     8881 ctgtcgaagc cgccgtactt cttagggtcc cagtccttct ttctggcgat cagcttatcg
     8941 ctgttcctct tgggcaggat agactctttg ctgaagccgc ctgtctgcac ctcggtcttt
     9001 ttcacgatat tcacttgggg catgctcagc actttccgca cggtggcaaa atcccggccc
     9061 ttatcccaca cgatctcccc ggtttcgccg tttgtctcga tcagaggccg cttccggatc
     9121 tcgccgttgg ccagggtaat ctcggtcttg aaaaagttca tgatgttgct gtagaagaag
     9181 tacttggcgg tagccttgcc gatttcctgc t
//
