LOCUS       sequence-373416-       11393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRISPRai cassette under EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11393)
  AUTHORS   Truong VA, Hsu MN, Kieu Nguyen NT, Lin MW, Shen CC, Lin CY, Hu YC
  TITLE     CRISPRai for simultaneous gene activation and inhibition to promote 
            stem cell chondrogenesis and calvarial bone regeneration.
  JOURNAL   Nucleic Acids Res. 2019 Jul 26;47(13):e74. doi: 10.1093/nar/gkz267.
  PUBMED    30997496
REFERENCE   2  (bases 1 to 11393)
  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: "Nucleic 
            Acids Res."; date: "2019-07-26"; volume: "47(13):e74. doi"; pages: "
            10.1093/nar/gkz267"
COMMENT     Sequence Label: pCRISPRai
FEATURES             Location/Qualifiers
     source          1..11393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    123..156
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        195..498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..1918
                     /label=rEF-1-alpha promoter
                     /note="strong constitutive promoter for rat elongation 
                     factor EF-1-alpha"
     misc_feature    955..1886
                     /label=rEF-1-alpha intron
                     /note="intron upstream of the start codon of rat 
                     EF-1-alpha"
     protein_bind    1951..1975
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1979..1988
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1985..2011,2012..2014,2015..4498,4499..4504,4505..4570,
                     4571..4573,4574..6088)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1985..2011,2012..2014,2015..4501,4502..4504,
                     4505..6088)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1985..6088
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6101..6121
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6122..6211
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             6227..6283
                     /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"
     regulatory      6311..6320
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6320..6706
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             6761..6781
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6797..7339
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             7364..7735
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             7763..7819
                     /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             7835..7855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8120..8305
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     primer_bind     complement(8383..8403)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8801..8812)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    9042..9176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9079..9098)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9133..9152
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    9218..9251
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(9395..9412)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9566..10154)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9646..9665)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10325..11116,11117..11185))
                     /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     10948..10967
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11186..11290)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(11365..11384)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
       61 aagctacaac aaggcaaggc ttgaccgaca attgtaagaa ttcatctccc ggtaccgcat
      121 gcataacttc gtataatgta tgctatacga agttatgcta gcaccatggc tcgagttctt
      181 aattaacctg caggcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      241 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      301 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      361 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      421 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      481 gtcatcgcta ttaccatgat gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      541 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttga
      601 ctagtggagc cgagagtaat tcatacaaaa ggagggatcg ccttcgcaag gggagagccc
      661 agggaccgtc cctaaattct cacagaccca aatccctgta gccgccccac gacagcgcga
      721 ggagcatgcg cccagggctg agcgcgggta gatcagagca cacaagctca cagtccccgg
      781 cggtgggggg aggggcgcgc tgagcggggg ccagggagct ggcgcggggc aaactgggaa
      841 agtggtgtcg tgtgctggct ccgccctctt cccgagggtg ggggagaacg gtatataagt
      901 gcggtagtcg ccttggacgt tctttttcgc aacgggtttg ccgtcagaac gcaggtgagt
      961 ggcgggtgtg gcttccgcgg gccccggagc tggagccctg ctctgagcgg gccgggctga
     1021 tatgcgagtg tcgtccgcag ggtttagctg tgagcattcc cacttcgagt ggcgggcggt
     1081 gcgggggtga gagtgcgagg cctagcggca accccgtagc ctcgcctcgt gtccggcttg
     1141 aggcctagcg tggtgtccgc cgccgcgtgc cactccggcc gcactatgcg ttttttgtcc
     1201 ttgctgccct cgattgcctt ccagcagcat gggctaacaa agggagggtg tggggctcac
     1261 tcttaaggag cccatgaagc ttacgttgga taggaatgga agggcaggag gggcgactgg
     1321 ggcccgcccg ccttcggagc acatgtccga cgccacctgg atggggcgag gcctgtggct
     1381 ttccgaagca atcgggcgtg agtttagcct acctgggcca tgtggcccta gcactgggca
     1441 cggtctggcc tggcggtgcc gcgttccctt gcctcccaac aagggtgagg ccgtcccgcc
     1501 cggcaccagt tgcttgcgcg gaaagatggc cgctcccggg gccctgttgc aaggagctca
     1561 aaatggagga cgcggcagcc cggtggagcg ggcgggtgag tcacccacac aaaggaagag
     1621 ggccttgccc ctcgccggcc gctgcttcct gtgaccccgt ggtctatcgg ccgcatagtc
     1681 acctcgggct tctcttgagc accgctcgtc gcggcggggg gaggggatct aatggcgttg
     1741 gagtttgttc acatttggtg ggtggagact agtcaggcca gcctggcgct ggaagtcatt
     1801 cttggaattt gcccctttga gtttggagcg aggctaattc tcaagcctct tagcggttca
     1861 aaggtatttt ctaaacccgt ttccaggtgt tgtgaaagcc accgctaatt caaagcaatc
     1921 tagcgtttaa acttaagctt ggtaccatca acaagtttgt acaaaaaagc aggctggcgc
     1981 caccatggac aagaagtact ccattgggct cgctatcggt accaacagcg tcggctgggc
     2041 cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga
     2101 tcgccacagc ataaagaaga acctcattgg agccctcctg ttcgactccg gggagacggc
     2161 cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat
     2221 ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca
     2281 taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt
     2341 tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag
     2401 gaagaagctg gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc
     2461 gcacatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag
     2521 cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt tcgaggagaa
     2581 cccgatcaac gcatccggcg ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc
     2641 ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg
     2701 taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc
     2761 cgaagatgcc aagctgcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct
     2821 ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc
     2881 cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc
     2941 tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt
     3001 cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta
     3061 cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat
     3121 cttggaaaaa atggacggca ccgaggagct gctggtaaag ctgaacagag aagatctgtt
     3181 gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact
     3241 gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa
     3301 gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg ctcggggaaa
     3361 ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga
     3421 ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga
     3481 taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac
     3541 agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt
     3601 cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt
     3661 taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga
     3721 aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa
     3781 aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat
     3841 tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta
     3901 cgctcatctc ttcgacgaca aagtcatgaa acagctcaag agacgccgat atacaggatg
     3961 ggggcggctg tcaagaaaac tgatcaatgg catccgagac aagcagagtg gaaagacaat
     4021 cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga
     4081 tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag
     4141 tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca
     4201 gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat
     4261 cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga
     4321 aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca
     4381 cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg
     4441 cagggacatg tacgtggatc aggaactgga catcaaccgg ttgtccgact acgacgtgga
     4501 tgctatcgtg ccccaaagct ttctcaaaga tgattctatt gataataaag tgttgacaag
     4561 atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat
     4621 gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa
     4681 tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag
     4741 gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat
     4801 gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa
     4861 gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa
     4921 caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa
     4981 aaaatatccc aagctggaat ctgaatttgt ttacggagac tataaagtgt acgatgttag
     5041 gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta
     5101 cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa
     5161 gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga
     5221 tttcgcgaca gtccgcaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga
     5281 agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct
     5341 gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt
     5401 cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag
     5461 cgtcaaggaa ctgctgggca tcacaatcat ggagcgatcc agcttcgaga aaaaccccat
     5521 cgactttctc gaagcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagctgcc
     5581 caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga
     5641 gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc
     5701 cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt
     5761 ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgagt tctccaaaag
     5821 agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga
     5881 taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg
     5941 cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac
     6001 aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag
     6061 aatcgacctc tctcagctcg gtggagacgg caccggcggg cccaagaaga agaggaaggt
     6121 atacccatac gatgttcctg actatgcggg ctatccctat gacgtcccgg actatgcagg
     6181 atcgtatcct tatgacgttc cagattacgc tggatctgga tctggcgcca ccaacttcag
     6241 cctgctgaag caggcaggcg acgtggaaga gaaccctggc cctggatctc tcgacgcgat
     6301 cgcacgtacg gccaccatgg cttcaaactt tactcagttc gtgctcgtgg acaatggtgg
     6361 gacaggggat gtgacagtgg ctccttctaa tttcgctaat ggggtggcag agtggatcag
     6421 ctccaactca cggagccagg cctacaaggt gacatgcagc gtcaggcagt ctagtgccca
     6481 gaagagaaag tataccatca aggtggaggt ccccaaagtg gctacccaga cagtgggcgg
     6541 agtcgaactg cctgtcgccg cttggaggtc ctacctgaac atggagctca ctatcccaat
     6601 tttcgctacc aattctgact gtgaactcat cgtgaaggca atgcaggggc tcctcaaaga
     6661 cggtaatcct atcccttccg ccatcgccgc taactcaggt atctacagcg ctggaggagg
     6721 tggaagcgga ggaggaggaa gcggaggagg aggtagcgga cctaagaaaa agaggaaggt
     6781 ggcggccgct ggatcccctt cagggcagat cagcaaccag gccctggctc tggcccctag
     6841 ctccgctcca gtgctggccc agactatggt gccctctagt gctatggtgc ctctggccca
     6901 gccacctgct ccagcccctg tgctgacccc aggaccaccc cagtcactga gcgctccagt
     6961 gcccaagtct acacaggccg gcgaggggac tctgagtgaa gctctgctgc acctgcagtt
     7021 cgacgctgat gaggacctgg gagctctgct ggggaacagc accgatcccg gagtgttcac
     7081 agatctggcc tccgtggaca actctgagtt tcagcagctg ctgaatcagg gcgtgtccat
     7141 gtctcatagt acagccgaac caatgctgat ggagtacccc gaagccatta cccggctggt
     7201 gaccggcagc cagcggcccc ccgaccccgc tccaactccc ctgggaacca gcggcctgcc
     7261 taatgggctg tccggagatg aagacttctc aagcatcgct gatatggact ttagtgccct
     7321 gctgtcacag atttcctcta gtgggcaggg aggaggtgga agcggcttca gcgtggacac
     7381 cagtgccctg ctggacctgt tcagcccctc ggtgaccgtg cccgacatga gcctgcctga
     7441 ccttgacagc agcctggcca gtatccaaga gctcctgtct ccccaggagc cccccaggcc
     7501 tcccgaggca gagaacagca gcccggattc agggaagcag ctggtgcact acacagcgca
     7561 gccgctgttc ctgctggacc ccggctccgt ggacaccggg agcaacgacc tgccggtgct
     7621 gtttgagctg ggagagggct cctacttctc cgaaggggac ggcttcgccg aggaccccac
     7681 catctccctg ctgacaggct cggagcctcc caaagccaag gaccccactg tctccgctgc
     7741 gatcgctgtc gagggatctg gcgccaccaa cttcagcctg ctgaagcagg caggcgacgt
     7801 ggaagagaac cctggccctg gatctaaaga ctttcccaaa aagaaaagaa aagtgggtag
     7861 tatgaaatca attcgctgta aaaactgcaa caaactgtta tttaaggcgg atagttttga
     7921 tcacattgaa atcaggtgtc cgcgttgcaa acgtcacatc ataatgctga atgcctgcga
     7981 gcatcccacg gagaaacatt gtgggaaaag agaaaaaatc acgcattctg acgaaaccgt
     8041 gcgttatggc tccactagtg ggcacaagct taatggcggt ggcggaggga tggatgctaa
     8101 gtcactaact gcctggtccc ggacactggt gaccttcaag gatgtatttg tggacttcac
     8161 cagggaggag tggaagctgc tggacactgc tcagcagatc gtgtacagaa atgtgatgct
     8221 ggagaactat aagaacctgg tttccttggg ttatcagctt actaagccag atgtgatcct
     8281 ccggttggag aagggagaag agccctagaa caattctaag gccttcgata atcaacctct
     8341 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     8401 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     8461 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     8521 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     8581 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     8641 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     8701 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
     8761 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     8821 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     8881 tcagacgagt cggatctccc tttgtcagta tcaagcttgt cgagaagtac tagaggatca
     8941 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     9001 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     9061 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     9121 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatctgat
     9181 cactgcttga gcctaggaga tccgaaccag cggccgcata acttcgtata atgtatgcta
     9241 tacgaagtta tctgcagtct cgacaagctt gttctagagc atgctgggga tgcggtgggc
     9301 tctatggctt ctgaggcgga aagaaccagc tgcattaatg aatcggccaa cgcgcgggga
     9361 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     9421 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     9481 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     9541 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     9601 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     9661 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     9721 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     9781 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     9841 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     9901 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     9961 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
    10021 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
    10081 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
    10141 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
    10201 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
    10261 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
    10321 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
    10381 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
    10441 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
    10501 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
    10561 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
    10621 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
    10681 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
    10741 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
    10801 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
    10861 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
    10921 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
    10981 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
    11041 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
    11101 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
    11161 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
    11221 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
    11281 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
    11341 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taa
//
