LOCUS       sequence-362425-        9954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pX459V2.0-HypaCas9 plasmid with mRuby2 reporter and TUBA1B sgRNA for
            N-terminal tagging of alpha-tubulin in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9954)
  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 9954)
  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: pX459-HypaCas9-mR2-TUBA1B_sgRNA
FEATURES             Location/Qualifiers
     source          1..9954
                     /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"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1235
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1247..1256
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1256..1321
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1328..1348
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1373..3442,3443..3445,3446..3448,3449..3454,3455..3460,
                     3461..3463,3464..5473)
                     /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             1373..5473
                     /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             5474..5521
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5537..5593
                     /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             5594..6304
                     /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             6314..6367
                     /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             6368..6967
                     /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(6368..6387)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     6864..6884
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(6995..7012)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7001..7208
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7217..7357
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7217..7346
                     /label=AAV2 ITR
     rep_origin      7432..7887
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7519..7538)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7729..7750
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7904..7923)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8023..8045
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8083..8101)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8169..8273
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8274..8342,8343..9134)
                     /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(8492..8511)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9305..9893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9794..9813
                     /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 gatgcactca cgctgcggga 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 gggggggggg gcgcgcgccr ggsggggsgg ggsggggsgr ggggsggggs ggggsgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc
     1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
     1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
     1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
     1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
     1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     3421 gtccgacggc ttcgccaaca gagccttcgc ggcgctgatc gccgacgaca gcctgacctt
     3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     5521 ggaattcggc agtggagcta ctaacttcag cctgctgaag caggctggag acgtggagga
     5581 gaaccctgga cctatggtgt ctaagggcga agagctgatc aaggaaaata tgcgtatgaa
     5641 ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa tgcacaggtg aaggagaagg
     5701 caatccgtac atgggaactc aaaccatgag gatcaaagtc atcgagggag gacccctgcc
     5761 atttgccttt gacattcttg ccacgtcgtt catgtatggc agccgtactt ttatcaagta
     5821 cccgaaaggc attcctgatt tctttaaaca gtcctttcct gagggtttta cttgggaaag
     5881 agttacgaga tacgaagatg gtggagtcgt caccgtcatg caggacacca gccttgagga
     5941 tggctgtctc gtttaccacg tccaagtcag aggggtaaac tttccctcca atggtcccgt
     6001 gatgcagaag aaaaccaagg gttgggagcc taatacagag atgatgtatc cagcagatgg
     6061 tggtctgagg ggatacactc atatggcact gaaagttgat ggtggtggcc atctgtcttg
     6121 ctctttcgta acaacttaca ggtcaaaaaa gaccgtcggg aacatcaaga tgcccggtat
     6181 ccatgccgtt gatcaccgcc tggaaaggtt agaggaaagt gacaatgaaa tgttcgtagt
     6241 acaacgcgaa cacgcagttg ccaagttcgc cgggcttggt ggtgggatgg acgagctgta
     6301 caagggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc
     6361 tggcccaatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     6421 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     6481 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     6541 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     6601 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     6661 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     6721 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
     6781 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     6841 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     6901 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     6961 tgcctgagaa ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     7021 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     7081 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     7141 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     7201 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     7261 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     7321 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     7381 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     7441 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     7501 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     7561 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     7621 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     7681 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     7741 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     7801 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     7861 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     7921 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     7981 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     8041 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     8101 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     8161 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     8221 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     8281 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     8341 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     8401 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     8461 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     8521 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     8581 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     8641 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     8701 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     8761 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     8821 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     8881 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     8941 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     9001 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     9061 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     9121 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     9181 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     9241 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     9301 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     9361 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     9421 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     9481 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     9541 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     9601 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     9661 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     9721 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     9781 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     9841 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     9901 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
