LOCUS       sequence-362416-        9951 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pX459V2.0 plasmid expressing HypaCas9 with P2A-mRuby2 and T2A-Puro, 
            with a sgRNA cloning site for CRISPR editing in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9951)
  AUTHORS   Husser MC, Ozugergin I, Resta T, Martin VJJ, Piekny AJ
  TITLE     Cytokinetic diversity in mammalian cells is revealed by the 
            characterization of endogenous anillin, Ect2 and RhoA.
  JOURNAL   Open Biol. 2022 Nov;12(11):220247. doi: 10.1098/rsob.220247. Epub 
            2022 Nov 23.
  PUBMED    36416720
REFERENCE   2  (bases 1 to 9951)
  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: "Open 
            Biol."; date: "2022-11"; volume: "12(11):220247. doi"; pages: " 
            10.1098/rsob.220247. Epub 2022 Nov 23"
COMMENT     Sequence Label: pX459V2.0-HypaCas9-mRuby2
FEATURES             Location/Qualifiers
     source          1..9951
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1325..1345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1370..3439,3440..3442,3443..3445,3446..3451,3452..3457,
                     3458..3460,3461..5470)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity without compromising on-target activity (Chen 
                     et al., 2017)"
                     /label=HypaCas9
                     /note="carries the mutations N692A, M694A, Q695A, and 
                     H698A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFAA
                     LIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1370..5470
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFAA
                     LIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5471..5518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5534..5590
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5591..6301
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             6311..6364
                     /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             6365..6964
                     /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(6365..6384)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6861..6881
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6992..7009)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6998..7205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7214..7354
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7214..7343
                     /label=AAV2 ITR
     rep_origin      7429..7884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7516..7535)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7726..7747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7901..7920)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8020..8042
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8080..8098)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8166..8270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8271..8339,8340..9131)
                     /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(8489..8508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9302..9890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9791..9810
                     /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 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccrggs ggggsggggs ggggsgrggg gsggggsggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
     1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     1381 cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga ttgccctgag
     2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     2821 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     3421 cgacggcttc gccaacagag ccttcgcggc gctgatcgcc gacgacagcc tgacctttaa
     3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     3901 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     4141 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga
     5521 attcggcagt ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa
     5581 ccctggacct atggtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt
     5641 ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa
     5701 tccgtacatg ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt
     5761 tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc
     5821 gaaaggcatt cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt
     5881 tacgagatac gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg
     5941 ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat
     6001 gcagaagaaa accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg
     6061 tctgagggga tacactcata tggcactgaa agttgatggt ggtggccatc tgtcttgctc
     6121 tttcgtaaca acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtatcca
     6181 tgccgttgat caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca
     6241 acgcgaacac gcagttgcca agttcgccgg gcttggtggt gggatggacg agctgtacaa
     6301 gggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg
     6361 cccaatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     6421 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     6481 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     6541 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     6601 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     6661 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     6721 ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc gaccaccagg gcaagggtct
     6781 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     6841 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     6901 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     6961 ctgagaattc taactagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     7021 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     7081 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     7141 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     7201 ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     7261 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     7321 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     7381 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     7441 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     7501 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     7561 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     7621 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     7681 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     7741 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     7801 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     7861 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     7921 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     7981 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     8041 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     8101 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     8161 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     8221 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     8281 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     8341 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     8401 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     8461 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     8521 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     8581 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     8641 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     8701 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     8761 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     8821 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     8881 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     8941 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     9001 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     9061 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     9121 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     9181 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     9241 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     9301 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     9361 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     9421 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     9481 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     9541 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     9601 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     9661 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     9721 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     9781 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     9841 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     9901 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
