LOCUS       sequence-457381-       10943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of His6-MBP-tagged Cas9-NLS-EGFP protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10943)
  AUTHORS   Burger A, Lindsay H, Felker A, Hess C, Anders C, Chiavacci E, Zaugg 
            J, Weber LM, Catena R, Jinek M, Robinson MD, Mosimann C
  TITLE     Maximizing mutagenesis with solubilized CRISPR-Cas9 
            ribonucleoprotein complexes.
  JOURNAL   Development. 2016 Apr 29. pii: dev.134809.
  PUBMED    27130213
REFERENCE   2  (bases 1 to 10943)
  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: 
            "Development. 2016 Apr 29. pii: dev.134809."
COMMENT     Sequence Label: pMJ922
FEATURES             Location/Qualifiers
     source          1..10943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2960..2969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2973..2993)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             complement(3048..4148)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(3051..3074)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(4158..4175)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4195..4217
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(4305..4324)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4306..4324)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4408..4427)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             5885..6076
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6070..6092)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    6178..6317
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6233..6252
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6332..6349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6503..7091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6583..6602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7262..8053,8054..8122))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7885..7904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8123..8227)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8295..8313
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(8340..8345,8346..8362,8363..8368)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(8734..8781)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     8777..8795
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(8927..8947)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(8954..9664,9665..9667))
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(8996..9017)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(9707..9727)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(9734..9754)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(9767..9793)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             complement(9773..9799)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(join(9803..10943,1..2963))
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(join(9803..10943,1..2963))
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 taggcgtcgt gggcgtggtg gtagttgttg atctcgcgca ctttgtaaaa ctggaaatcc
       61 ttccggaaat cggacaccag cttggacttc agggtgatca ctttcacttc ccggatcagt
      121 ttgtcgttct cgtcgtactt agtgttcatc cgggagtcca ggatctgtgc cacgtgcttt
      181 gtgatctgcc gggtttccac cagctgtctc ttgatgaagc cggccttatc cagttcgctc
      241 aggccgcctc tctcggcctt ggtcagattg tcgaacttcc tctgggtaat cagcttggca
      301 ttcagcagct ggcgccagta gttcttcatc ttcttcacga cctcttcgga gggcacgttg
      361 tcgctcttgc cccggttctt gtcgctccga gtcagcactt tgttatcgat ggagtcgtcc
      421 ttcagaaagc tctgaggcac gatatggtcc acatcgtagt cggacagccg gttgatgtcc
      481 agttcctggt ccacgtacat atcccgccca ttctgcaggt agtacaggta cagcttctcg
      541 ttctgcagct gggtgttttc cacggggtgt tctttcagga tctggctgcc cagctctttg
      601 atgccctctt cgatccgctt cattctctcg cggctgttct tctgtccctt ctgggtggtc
      661 tggttctctc tggccatttc gatcacgatg ttctcgggct tgtgccggcc catcactttc
      721 acgagctcgt ccaccacctt cactgtctgc aggatgccct tcttaatggc ggggctgccg
      781 gccagattgg caatgtgctc gtgcaggcta tcgccctggc cggacacctg ggctttctgg
      841 atgtcctctt taaaggtcag gctgtcgtcg tggatcagct gcatgaagtt tctgttggcg
      901 aagccgtcgg acttcaggaa atccaggatt gtcttgccgg actgcttgtc ccggatgccg
      961 ttgatcagct tccggctcag cctgccccag ccggtgtatc tccgccgctt cagctgcttc
     1021 atcactttgt cgtcgaacag gtgggcatag gttttcagcc gttcctcgat catctctctg
     1081 tcctcaaaca gtgtcagggt cagcacgata tcttccagaa tgtcctcgtt ttcctcattg
     1141 tccaggaagt ccttgtcctt gataattttc agcagatcgt ggtatgtgcc cagggaggcg
     1201 ttgaaccgat cttccacgcc ggagatttcc acggagtcga agcactcgat tttcttgaag
     1261 tagtcctctt tcagctgctt cacggtcact ttccggttgg tcttgaacag caggtccacg
     1321 atggcttttt tctgctcgcc gctcaggaag gcgggctttc tcattccctc ggtcacgtat
     1381 ttcactttgg tcagctcgtt gtacacggtg aagtactcgt acagcaggct gtgcttgggc
     1441 agcaccttct cgttgggcag gttcttatcg aagttggtca tccgctcgat gaagctctgg
     1501 gcgctggcgc ccttgtccac cacttcctcg aagttccagg gggtgatggt ttcctcgctc
     1561 tttctggtca tccaggcgaa tctgctgttt cccctggcca gagggcccac gtagtagggg
     1621 atgcggaagg tcaggatctt ctcgatcttt tcccggttgt ccttcaggaa tgggtaaaaa
     1681 tcttcctgcc gccgcagaat ggcgtgcagc tctcccaggt ggatctggtg ggggatgctg
     1741 ccgttgtcga aggtccgctg cttccgcagc aggtcctctc tgttcagctt cacgagcagt
     1801 tcctcggtgc cgtccatctt ttccaggatg ggcttgatga acttgtagaa ctcttcctgg
     1861 ctggctccgc catcgatgta gccggcgtag ccgttcttgc tctggtcgaa gaaaatctct
     1921 ttgtacttct caggcagctg ctgccgcacg agagctttca gcagggtcag gtcctggtgg
     1981 tgctcgtcgt atctcttgat catagaggcg ctcagggggg ccttggtgat ctcggtgttc
     2041 actctcagga tgtcgctcag caggatggcg tcggacaggt tcttggcggc cagaaacagg
     2101 tcggcgtact ggtcgccgat ctgggccagc aggttgtcca ggtcgtcgtc gtaggtgtcc
     2161 ttgctcagct gcagtttggc atcctcggcc aggtcgaagt tgctcttgaa gttgggggtc
     2221 aggcccaggc tcagggcaat caggttgccg aacaggccat tcttcttctc gccgggcagc
     2281 tgggcgatca gattttccag ccgtctgctc ttgctcagtc tggcagacag gatggccttg
     2341 gcgtccacgc cgctggcgtt gatggggttt tcctcgaaca gctggttgta ggtctgcacc
     2401 agctggatga acagcttgtc cacgtcgctg ttgtcggggt tcaggtcgcc ctcgatcagg
     2461 aagtggcccc ggaacttgat catgtgggcc agggccagat agatcagccg caggtcggcc
     2521 ttgtcggtgc tgtccaccag tttctttctc aggtggtaga tggtggggta cttctcgtgg
     2581 taggccacct cgtccacgat gttgccgaag atggggtgcc gctcgtgctt cttatcctct
     2641 tccaccagga aggactcttc cagtctgtgg aagaagctgt cgtccacctt ggccatctcg
     2701 ttgctgaaga tctcttgcag atagcagatc cggttcttcc gtctggtgta tcttcttctg
     2761 gcggttctct tcagccgggt ggcctcggct gtttctccgc tgtcgaacag cagggcgccg
     2821 atcaggttct tcttgatgct gtgccggtcg gtgttgccca gcaccttgaa tttcttgctg
     2881 ggcaccttgt actcgtcggt gatcacggcc cagcccacag agttggtgcc gatgtccagg
     2941 ccgatgctgt acttcttgtc catggtggca ttggattgga agtacaggtt ttcctcgatc
     3001 ccgaggttgt tgttattgtt attgttgttg ttgttcgagc tcgaattagt ctgcgcgtct
     3061 ttcagggctt catcgacagt ctgacgaccg ctggcggcgt tgatcaccgc agtacgcacg
     3121 gcataccaga aagcggacat ctgcgggatg ttcggcatga tttcaccttt ctgggcgttt
     3181 tccatagtgg cggcaatacg tggatctttc gccaactctt cctcgtaaga cttcagcgct
     3241 acggcaccca gcggtttgtc tttattaacc gcttccagac cttcatcagt cagcagatag
     3301 ttttcgagga actcttttgc cagctctttg ttcggactgg cggcgttaat acctgcgctc
     3361 agcacgccaa cgaacggttt ggatggttga cccttgaagg tcggcagtac cgttacacca
     3421 taattcactt tgctggtgtc gatgttggac catgcccacg ggccgttgat ggtcatcgct
     3481 gtttcgcctt tattaaaggc agcttctgcg atggagtaat cggtgtctgc attcatgtgt
     3541 ttgtttttaa tcaggtcaac caggaaggtc agacccgctt tcgcgccagc gttatccacg
     3601 cccacgtctt taatgtcgta cttgccgttt tcatacttga acgcataacc cccgtcagca
     3661 gcaatcagcg gccaggtgaa gtacggttct tgcaggttga acatcagcgc gctcttacct
     3721 ttcgctttca gttctttatc cagcgccggg atctcttccc aggtttttgg cgggttcggc
     3781 agcagatctt tgttataaat cagcgataac gcttcaacag cgatcgggta agcaatcagc
     3841 ttgccgttgt aacgtacggc atcccaggta aacggataca gcttgtcctg gaacgctttg
     3901 tccggggtga tttcagccaa caggccagat tgagcgtagc caccaaagcg gtcgtgtgcc
     3961 cagaagataa tgtcagggcc atcgccagtt gccgcaacct gtgggaattt ctcttccagt
     4021 ttatccggat gctcaacggt gactttaatt ccggtatctt tctcgaattt cttaccgact
     4081 tcagcgagac cgttatagcc tttatcgccg ttaatccaga ttaccagttt accttcttcg
     4141 attttcatag aagaaccatg gtgatggtga tggtgagaag atttcatatg tatatctcct
     4201 tcttaaagtt aaacaaaatt atttctagaa ctcgagtcct gcagggcggc cgcttaatta
     4261 aagagctctc cggagccggc actagaggga aaccgttgtg gtctccctat agtgagtcgt
     4321 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     4381 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     4441 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     4501 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     4561 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     4621 gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca
     4681 tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
     4741 cggcagcgct ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg
     4801 gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc
     4861 ccgccaccaa acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc
     4921 tgggctacgt cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc
     4981 ttctcgcttc cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag
     5041 atgacgacca tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga
     5101 tcattggacc gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt
     5161 tggcatggat tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg
     5221 catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac
     5281 cactccaaga attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc
     5341 ttggcagaac atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg
     5401 cagcgttggg tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     5461 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     5521 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     5581 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     5641 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     5701 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     5761 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     5821 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     5881 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     5941 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     6001 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     6061 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     6121 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     6181 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     6241 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     6301 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     6361 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     6421 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6481 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6541 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6601 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6661 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6721 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6781 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6841 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6901 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     6961 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7021 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     7081 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     7141 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     7201 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     7261 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     7321 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7381 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7441 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7501 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7561 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7621 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7681 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7741 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7801 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7861 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7921 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7981 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     8041 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     8101 cacggaaatg ttgaatactc atactcttcc tttttcaatg ttattgaagc atttatcagg
     8161 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     8221 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     8281 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa ttctcatgtt
     8341 tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa ttgctaacgc
     8401 agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc
     8461 ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc
     8521 gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata tgcgttgatg
     8581 caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg ccgcccagtc
     8641 ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac cacacccgtc
     8701 ctgtggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga
     8761 ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact cagcttcctt
     8821 tcgggctttg ttagcagccg gatcagcgcg cacgcgtggt accggccggc caccaggtgg
     8881 cgcgccgcga tcgcggatcc gttatccact tccaatgtta ttactcaact tttcgttttt
     8941 tcttaggtga ccccttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     9001 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagc ttggactggg
     9061 tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     9121 ggtagtggtc ggcgagctgc acgctgccgt cctcgacgtt gtggcggatc ttgaagttcg
     9181 ccttgatgcc gttcttctgc ttgtcggccg tgatatagac gttgtggctg ttgaagttgt
     9241 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     9301 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg taggtgccgt
     9361 cgtccttgaa ggagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
     9421 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
     9481 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
     9541 tcagcttgcc gttggtggca tcgccctcgc cctcgccgcg cacgctgaac ttgtggccgt
     9601 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     9661 tgctcacgga tccgctgctg ccgttgctcc cactaccaat gccgtccact ttccgctttt
     9721 tcttaggatc ttccaccttg cgttttttct tgggggagcc gctgcccagg ctggcataat
     9781 cgggcacgtc atagggatag gcgtcgcctc ccagctgaga caggtcgatc cgtgtctcgt
     9841 acaggccggt gatgctctgg tggatcaggg tggcgtccag cacctctttg gtgctggtgt
     9901 acctcttccg gtcgatggtg gtgtcaaagt acttgaaggc ggcaggggct cccagattgg
     9961 tcagggtaaa caggtggatg atattctcgg cctgctctct gataggcttg tctctgtgct
    10021 tgttgtaggc gctcagcacc ttgtccagat tagcgtcggc caggatcact ctcttggaga
    10081 actcgctgat ctgctcgatg atctcgtcca ggtagtgttt gtgctgttcc acaaacagct
    10141 gtttctgctc attatcctcg ggggagccct tcagcttctc atagtggctg gccaggtaca
    10201 ggaagttcac atatttggag ggcagggcca gttcgtttcc cttctgcagt tcgccggcag
    10261 aggccagcat tctcttccgg ccgttttcca gctcgaacag ggagtactta ggcagcttga
    10321 tgatcaggtc ctttttcact tctttgtagc ccttggcttc cagaaagtcg atgggattct
    10381 tctcgaagct gcttctttcc atgatggtga tccccagcag ctctttcaca ctcttcagtt
    10441 tcttggactt gcccttttcc actttggcca ccaccagcac agaataggcc acggtggggc
    10501 tgtcgaagcc gccgtacttc ttagggtccc agtccttctt tctggcgatc agcttgtcgc
    10561 tgttcctctt gggcaggata gactctttgc tgaagccgcc tgtctgcacc tcggtctttt
    10621 tcacgatatt cacttggggc atagacagca ctttccgcac ggtggcaaag tcccggccct
    10681 tatcccacac gatctcgcct gtttcgccgt ttgtctcgat cagaggccgc ttccggatct
    10741 cgccgttggc cagggtaatc tcggtcttga aaaagttcat gatgttgctg tagaagaagt
    10801 acttggcggt agccttgccg atttcctgct cgctcttggc gatcatcttc cgcacgtcgt
    10861 acaccttgta gtcgccgtac acgaactcgc tttccagctt agggtacttt ttgatcaggg
    10921 cggttcccac gacggcgttc agg
//
