LOCUS       sequence-482293-        9292 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Mammalian expression of SpCas9 and gRNA targeting ARHGEF7 
            (beta-Pix).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9292)
  AUTHORS   Weiss BG, Keth JM, Bhatt K, Doyal M, Hahn KM, Noh J, Isogai T, 
            Danuser G
  TITLE     Morphological control of merlin-Rac antagonism in 
            proliferation-promoting signaling.
  JOURNAL   Sci Signal. 2025 May 20;18(887):eadk0922. doi: 
            10.1126/scisignal.adk0922. Epub 2025 May 20.
  PUBMED    40392939
REFERENCE   2  (bases 1 to 9292)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci 
            Signal."; date: "2025-05-20"; volume: "18(887):eadk0922. doi"; 
            pages: " 10.1126/scisignal.adk0922. Epub 2025 May 20"
COMMENT     Sequence Label: pX458-ARHGEF7/B-Pix
FEATURES             Location/Qualifiers
     source          1..9292
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        651..899
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     651..671
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     822..841
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        921..996
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1093..1378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1659..1886
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1898..1907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1907..1972
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1979..1999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2024..6124
                     /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             2024..6124
                     /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             6125..6172
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6188..6241
                     /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             join(6242..6244,6245..6955)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6242..6244,6245..6955)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6545..6564)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6892..6913
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6983..7000)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6989..7196
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7205..7345
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7205..7334
                     /label=AAV2 ITR
     rep_origin      7420..7875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7507..7526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7717..7738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7892..7911)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8011..8033
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8071..8089)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8157..8261
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8262..8330,8331..9122)
                     /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(8480..8499)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
      661 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
      721 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
      781 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
      841 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
      901 acctcgcgca cgtagttgct gttttagagc tagaaatagc aagttaaaat aaggctagtc
      961 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata
     1021 gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc
     1081 tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1141 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     1201 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     1261 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     1321 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     1381 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     1441 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     1501 gcgcgccccg gggggggggg gggggggggg aggggcgggg cggggcgagg cggagaggtg
     1561 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     1621 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1681 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1741 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
     1801 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
     1861 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
     1921 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     1981 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     2041 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     2101 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     2161 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     2221 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     2281 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     2341 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     2401 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     2461 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     2521 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     2581 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     2641 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     2701 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     2761 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     2821 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     2881 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     2941 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     3001 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     3061 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     3121 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     3181 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     3241 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     3301 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     3361 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     3421 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     3481 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     3541 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     3601 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     3661 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     3721 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     3781 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     3841 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     3901 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     3961 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     4021 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     4081 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     4141 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     4201 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     4261 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     4321 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     4381 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     4441 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     4501 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     4561 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     4621 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     4681 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     4741 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     4801 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     4861 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     4921 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     4981 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     5041 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     5101 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     5161 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     5221 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     5281 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     5341 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     5401 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     5461 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     5521 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     5581 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     5641 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     5701 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     5761 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     5821 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     5881 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     5941 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     6001 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     6061 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     6121 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg
     6181 cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     6241 agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     6301 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     6361 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     6421 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     6481 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     6541 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     6601 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     6661 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     6721 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     6781 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     6841 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     6901 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggaatt
     6961 ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     7021 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     7081 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     7141 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg
     7201 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     7261 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     7321 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     7381 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     7441 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     7501 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     7561 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     7621 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     7681 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     7741 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     7801 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     7861 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     7921 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     7981 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     8041 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     8101 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     8161 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     8221 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     8281 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     8341 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     8401 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     8461 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     8521 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     8581 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     8641 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     8701 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     8761 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     8821 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     8881 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     8941 ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac
     9001 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     9061 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     9121 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     9181 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     9241 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
