LOCUS       sequence-273809-       11687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutisite Gateway mediated vector construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11687)
  AUTHORS   Yoshimatsu S, Sone T, Nakajima M, Sato T, Okochi R, Ishikawa M, 
            Nakamura M, Sasaki E, Shiozawa S, Okano H
  TITLE     A versatile toolbox for knock-in gene targeting based on the 
            Multisite Gateway technology.
  JOURNAL   PLoS One. 2019 Aug 27;14(8):e0221164. doi: 
            10.1371/journal.pone.0221164. eCollection 2019.
  PUBMED    31454364
REFERENCE   2  (bases 1 to 11687)
  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: "PLoS One.";
            date: "2019-08-27"; volume: "14(8):e0221164. doi"; pages: " 
            10.1371/journal.pone.0221164. eCollection 2019"
COMMENT     Sequence Label: pDEST-Cas9-tdTomato
FEATURES             Location/Qualifiers
     source          1..11687
                     /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..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1245..1254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1254..1319
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1326..1346
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1371..5471
                     /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             1371..5471
                     /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             5472..5519
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5535..5588
                     /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             5589..7013
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
                     VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
                     ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
                     LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN
                     FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL
                     KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER
                     SEGRHHLFLYGMDELYK"
     primer_bind     complement(7041..7058)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7047..7254
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7257..7273
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7314..7437
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(7462..7467,7468..7485,7486..7492)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             7546..8226
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(7612..7631)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             8546..8851
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     8730..8749
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(8892..9015)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(9077..9093)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9077..9093)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      9165..9620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9252..9271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9462..9483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9637..9656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9756..9778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9816..9834)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9902..10006
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10007..10075,10076..10867)
                     /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(10225..10244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11038..11626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11527..11546
                     /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 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
     1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
     1381 acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga
     2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
     3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg
     5521 aattcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     5581 ctggcccagt gagcaagggc gaggaggtca tcaaagagtt catgcgcttc aaggtgcgca
     5641 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     5701 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct
     5761 gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag caccccgccg
     5821 acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     5881 acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag gacggcacgc
     5941 tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc gtaatgcaga
     6001 aaaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga
     6061 agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
     6121 agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac gtggacacca
     6181 agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgctccg
     6241 agggccgcca ccacctgttc ctggggcatg gcaccggcag caccggcagc ggcagctccg
     6301 gcaccgcctc ctccgaggac aacaacatgg ccgtcatcaa agagttcatg cgcttcaagg
     6361 tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     6421 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct
     6481 tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc
     6541 ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg
     6601 tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg
     6661 gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa
     6721 tgcagaaaaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg
     6781 tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg
     6841 agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg
     6901 acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc
     6961 gctccgaggg ccgccaccac ctgttcctgt acggcatgga cgagctgtac aaggaattct
     7021 aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     7081 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     7141 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     7201 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcgtaa
     7261 aacgacggcc agtgccaagc ttgcatgcct gcaggtcggg atatctgaat tatcaacttt
     7321 gtataataaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
     7381 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatggcg
     7441 gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtggatt
     7501 ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
     7561 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
     7621 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
     7681 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
     7741 ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct ggtgatatgg
     7801 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
     7861 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
     7921 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc
     7981 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
     8041 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
     8101 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
     8161 aatgaattac aacagtactg cgatgagtgg caggcggggc gtaatctaga ggatccggct
     8221 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
     8281 atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc agcgtattac
     8341 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc
     8401 ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa
     8461 gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt
     8521 gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata aaagagagag
     8581 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     8641 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     8701 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     8761 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     8821 cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc
     8881 tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt
     8941 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcaactt
     9001 ttctatacaa agttgatagc ttgatatcaa tgactctaga ggatccccgg gtaccgagct
     9061 cgaattcgta atcatggtca tagctgtttc ctggcctgat gcggtatttt ctccttacgc
     9121 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     9181 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     9241 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     9301 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     9361 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     9421 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     9481 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     9541 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     9601 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     9661 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     9721 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     9781 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     9841 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     9901 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     9961 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
    10021 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
    10081 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
    10141 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
    10201 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
    10261 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
    10321 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
    10381 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
    10441 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
    10501 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
    10561 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
    10621 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
    10681 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
    10741 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
    10801 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
    10861 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
    10921 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
    10981 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
    11041 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
    11101 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
    11161 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
    11221 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
    11281 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
    11341 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
    11401 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
    11461 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
    11521 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
    11581 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
    11641 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
