LOCUS       sequence-471729-        9245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 from S. pyogenes with 2A-mCherry, gRNA to knock-in msfGFP to 
            EWSR1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9245)
  AUTHORS   Zhang Y, Stoppelkamp I, Fernandez-Pernas P, Allram M, Charman M, 
            Magalhaes AP, Piedavent-Salomon M, Sommer G, Sung YC, Meyer K, Grams
            N, Halko E, Dongre S, Meierhofer D, Malszycki M, Ilik IA, Aktas T, 
            Kraushar ML, Vastenhouw N, Weitzman MD, Grebien F, Niskanen H, Hnisz
            D
  TITLE     Probing condensate microenvironments with a micropeptide killswitch.
  JOURNAL   Nature. 2025 Jun 4. doi: 10.1038/s41586-025-09141-5.
  PUBMED    40468084
REFERENCE   2  (bases 1 to 9245)
  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: "Nature. 
            2025 Jun 4. doi: 10.1038/s41586-025-09141-5."
COMMENT     Sequence Label: pX458_mCherry-EWSR1-gRNA
FEATURES             Location/Qualifiers
     source          1..9245
                     /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          965..1192
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1204..1213
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1213..1278
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1285..1305
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1330..5430
                     /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             1330..5430
                     /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             5431..5478
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5494..5547
                     /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             5554..6258
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(5696..5714)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6286..6303)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6292..6499
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6508..6648
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6508..6637
                     /label=AAV2 ITR
     rep_origin      6723..7178
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6810..6829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7020..7041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7195..7214)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7314..7336
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7374..7392)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7460..7564
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7565..7633,7634..8425)
                     /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(7783..7802)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8596..9184
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9085..9104
                     /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 tggtggaact gcggtcgcgc 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 ggggcgaggg
      841 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      901 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      961 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     1021 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     1081 tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg
     1141 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc
     1201 ggtgccacca tggactataa ggaccacgac ggagactaca aggatcatga tattgattac
     1261 aaagacgatg acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc
     1321 ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg
     1381 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     1441 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     1501 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     1561 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     1621 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     1681 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     1741 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     1801 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     1861 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     1921 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     1981 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     2041 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     2101 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     2161 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     2221 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     2281 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     2341 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     2401 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     2461 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     2521 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     2581 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     2641 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     2701 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     2761 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc
     2821 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     2881 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     2941 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     3001 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     3061 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     3121 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     3181 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     3241 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     3301 tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca
     3361 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac
     3421 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     3481 agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg
     3541 cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac
     3601 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc
     3661 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     3721 caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat
     3781 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     3841 gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc
     3901 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     3961 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     4021 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     4081 agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg
     4141 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     4201 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     4261 aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc
     4321 aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg
     4381 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     4441 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     4501 aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     4561 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     4621 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     4681 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     4741 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     4801 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     4861 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4921 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4981 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     5041 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     5101 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     5161 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     5221 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     5281 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     5341 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     5401 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     5461 caggcaaaaa agaaaaagga attcggcagt ggagagggca gaggaagtct gctaacatgc
     5521 ggtgacgtcg aggagaatcc tggcccagaa ttcgtgagca agggcgagga ggataacatg
     5581 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     5641 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     5701 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     5761 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     5821 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     5881 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     5941 ggcaccaact tcccctccga cggccccgta atgcagaaga aaaccatggg ctgggaggcc
     6001 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     6061 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     6121 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     6181 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     6241 atggacgagc tgtacaagga attctaacta gagctcgctg atcagcctcg actgtgcctt
     6301 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     6361 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     6421 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga
     6481 atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct
     6541 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     6601 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     6661 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     6721 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6781 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6841 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6901 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     6961 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     7021 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     7081 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     7141 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     7201 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     7261 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     7321 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     7381 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     7441 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     7501 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     7561 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     7621 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     7681 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     7741 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     7801 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     7861 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     7921 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     7981 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     8041 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     8101 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     8161 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     8221 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     8281 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     8341 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     8401 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     8461 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     8521 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     8581 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     8641 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     8701 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     8761 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     8821 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     8881 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     8941 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     9001 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     9061 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     9121 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     9181 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     9241 catgt
//
