LOCUS       sequence-494460-        9179 bp    DNA     circular SYN 02-DEC-2025
DEFINITION  CRISPR vector co-expressing Cas9 and a mouse Fzd1/2 gRNA (conserved 
            region).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9179)
  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 9179)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 2, 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-Fzd1/2 gRNA
FEATURES             Location/Qualifiers
     source          1..9179
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1010..1237
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1249..1258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1258..1323
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1330..1350
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1375..5475
                     /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             1375..5475
                     /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             5476..5523
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5539..5592
                     /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             5593..6192
                     /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(5593..5612)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6089..6109
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6220..6237)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6226..6433
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6442..6582
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6442..6571
                     /label=AAV2 ITR
     rep_origin      6657..7112
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6744..6763)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6954..6975
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7129..7148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7248..7270
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7308..7326)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7394..7498
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7499..7567,7568..8359)
                     /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(7717..7736)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8530..9118
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9019..9038
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gatgctctac ttcttcagca gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggc gcgcgccggg cggggggggg gggggggggg gggggggggg gggggggggg
      901 gggggggggg gcgggggggg gcgggggggg gggggggcgc ccagtgtcgc tttgttgggg
      961 gtgggggggg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     1021 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1081 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     1141 ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa
     1201 ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac
     1261 tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat
     1321 aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag
     1381 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac
     1441 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
     1501 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
     1561 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
     1621 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     1681 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     1741 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     1801 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     1861 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     1921 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     1981 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     2041 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc
     2101 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     2161 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     2221 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     2281 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     2341 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     2401 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     2461 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     2521 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     2581 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     2641 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     2701 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     2761 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     2821 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     2881 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     2941 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     3001 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     3061 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     3121 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     3181 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     3241 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     3301 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     3361 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     3421 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     3481 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     3541 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     3601 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     3661 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     3721 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     3781 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     3841 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct
     3901 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     3961 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     4021 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     4081 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     4141 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     4201 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     4261 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     4321 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     4381 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     4441 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     4501 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     4561 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     4621 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     4681 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     4741 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     4801 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     4861 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     4921 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     4981 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     5041 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     5101 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     5161 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     5221 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     5281 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     5341 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     5401 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     5461 cagctgggag gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa
     5521 aaggaattcg gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     5581 aatcctggcc caatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     5641 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     5701 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     5761 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     5821 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     5881 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     5941 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     6001 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     6061 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     6121 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     6181 cccggtgcct gagaattcta actagagctc gctgatcagc ctcgactgtg ccttctagtt
     6241 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     6301 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     6361 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     6421 ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     6481 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     6541 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     6601 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     6661 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6721 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6781 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6841 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     6901 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6961 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     7021 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     7081 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     7141 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     7201 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     7261 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     7321 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     7381 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     7441 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     7501 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     7561 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     7621 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     7681 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     7741 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     7801 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     7861 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     7921 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     7981 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     8041 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     8101 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     8161 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     8221 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     8281 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     8341 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     8401 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     8461 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     8521 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     8581 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     8641 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     8701 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     8761 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     8821 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     8881 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     8941 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     9001 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     9061 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     9121 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
