LOCUS       Exported               11300 bp DNA     circular SYN 06-APR-2022
DEFINITION  Expresses Cas9 nuclease and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pX330A-1x2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11300)
  AUTHORS   Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T
  TITLE     Multiplex genome engineering in human cells using all-in-one 
            CRISPR/Cas9 vector system.
  JOURNAL   Sci Rep. 2014 Jun 23;4:5400. doi: 10.1038/srep05400.
  PUBMED    24954249
REFERENCE   2  (bases 1 to 11300)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 16, 2022 from SnapGene Viewer 6.0.4
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2014-06-23"; volume: "4:5400. doi"; pages: " 
            10.1038/srep05400"
FEATURES             Location/Qualifiers
     source          1..11300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        295..572
                     /label=chicken beta-actin promoter
     intron          573..800
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      812..821
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             821..886
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             893..913
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(893..913)
                     /label=Cas9_BamHI_Primer R2
     CDS             938..5038
                     /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             5039..5086
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5065..5087)
                     /label=Cas9_BamHI_Primer R
     misc_feature    5087..5122
                     /label=SG3-linker
     CDS             5123..7813
                     /codon_start=1
                     /label=T4 DNA polymerase
                     /translation="KEFYISIETVGNNIVERYIDENGKERTREVEYLPTMFRHCKEESK
                     YKDIYGKNCAPQKFPSMKDARDWMKRMEDIGLEALGMNDFKLAYISDTYGSEIVYDRKF
                     VRVANCDIEVTGDKFPDPMKAEYEIDAITHYDSIDDRFYVFDLLNSMYGSVSKWDAKLA
                     AKLDCEGGDEVPQEILDRVIYMPFDNERDMLMEYINLWEQKRPAIFTGWNIEGFDVPYI
                     MNRVKMILGERSMKRFSPIGRVKSKLIQNMYGSKEIYSIDGVSILDYLDLYKKFAFTNL
                     PSFSLESVAQHETKKGKLPYDGPINKLRETNHQRYISYNIIDVESVQAIDKIRGFIDLV
                     LSMSYYAKMPFSGVMSPIKTWDAIIFNSLKGEHKVIPQQGSHVKQSFPGAFVFEPKPIA
                     RRYIMSFDLTSLYPSIIRQVNISPETIRGQFKVHPIHEYIAGTAPKPSDEYSCSPNGWM
                     YDKHQEGIIPKEIAKVFFQRKDWKKKMFAEEMNAEAIKKIIMKGAGSCSTKPEVERYVK
                     FSDDFLNELSNYTESVLNSLIEECEKAATLANTNQLNRKILINSLYGALGNIHFRYYDL
                     RNATAITIFGQVGIQWIARKINEYLNKVCGTNDEDFIAAGDTDSVYVCVDKVIEKVGLD
                     RFKEQNDLVEFMNQFGKKKMEPMIDVAYRELCDYMNNREHLMHMDREAISCPPLGSKGV
                     GGFWKAKKRYALNVYDMEDKRFAEPHLKIMGMETQQSSTPKAVQEALEESIRRILQEGE
                     ESVQEYYKNFEKEYRQLDYKVIAEVKTANDIAKYDDKGWPGFKCPFHIRGVLTYRRAVS
                     GLGVAPILDGNKVMVLPLREGNPFGDKCIAWPSGTELPKEIRSDVLSWIDHSTLFQKSF
                     VKPLAGMCESAGMDYEEKASLDFLFG"
     primer_bind     complement(7813..7846)
                     /label=Cas9_BamHI_Primer R
     primer_bind     complement(7823..7876)
                     /label=Cas9_BamHI_Primer R2
     CDS             7823..7843
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7866..7883)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7872..8079
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     7872..7889
                     /label=bGH_poly_seq_F
     primer_bind     8046..8068
                     /label=wo_sgRNA_primer_F
     repeat_region   8088..8228
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8088..8217
                     /label=AAV2 ITR
     rep_origin      8303..8758
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8390..8409)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8600..8621
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8775..8794)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8894..8916
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8954..8972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9040..9144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             9145..10005
                     /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(9363..9382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10176..10764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10665..10684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(10839..11144)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     10994..11016
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     11008..11025
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     11009..11025
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     11035..11054
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        11035..11053
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(11101..11121)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(11101..11119)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11140..11156)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11140..11156)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11153..11175)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11164..11180
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(11188..11218)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11233..11254
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      421 cgccaggcgg ggcggggcgg ggcggggggc ggggcggggc gaggcggaga ggtgcggcgg
      481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      601 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      661 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagctg agcaagaggt
      721 aagggtttaa gggatggttg gttggtgggg tattaatgtt taattacctg gagcacctgc
      781 ctgaaatcac tttttttcag gttggaccgg tgccaccatg gactataagg accacgacgg
      841 agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg ccccaaagaa
      901 gaagcggaag gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct
      961 ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
     1021 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
     1081 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga gaaccgccag
     1141 aagaagatac accagacgga agaaccggat ctgctatctg caagagatct tcagcaacga
     1201 gatggccaag gtggacgaca gcttcttcca cagactggaa gagtccttcc tggtggaaga
     1261 ggataagaag cacgagcggc accccatctt cggcaacatc gtggacgagg tggcctacca
     1321 cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
     1381 cgacctgcgg ctgatctatc tggccctggc ccacatgatc aagttccggg gccacttcct
     1441 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca tccagctggt
     1501 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa
     1561 ggccatcctg tctgccagac tgagcaagag cagacggctg gaaaatctga tcgcccagct
     1621 gcccggcgag aagaagaatg gcctgttcgg aaacctgatt gccctgagcc tgggcctgac
     1681 ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
     1741 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
     1801 gtttctggcc gccaagaacc tgtccgacgc catcctgctg agcgacatcc tgagagtgaa
     1861 caccgagatc accaaggccc ccctgagcgc ctctatgatc aagagatacg acgagcacca
     1921 ccaggacctg accctgctga aagctctcgt gcggcagcag ctgcctgaga agtacaaaga
     1981 gattttcttc gaccagagca agaacggcta cgccggctac attgacggcg gagccagcca
     2041 ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
     2101 gctcgtgaag ctgaacagag aggacctgct gcggaagcag cggaccttcg acaacggcag
     2161 catcccccac cagatccacc tgggagagct gcacgccatt ctgcggcggc aggaagattt
     2221 ttacccattc ctgaaggaca accgggaaaa gatcgagaag atcctgacct tccgcatccc
     2281 ctactacgtg ggccctctgg ccaggggaaa cagcagattc gcctggatga ccagaaagag
     2341 cgaggaaacc atcaccccct ggaacttcga ggaagtggtg gacaagggcg cttccgccca
     2401 gagcttcatc gagcggatga ccaacttcga taagaacctg cccaacgaga aggtgctgcc
     2461 caagcacagc ctgctgtacg agtacttcac cgtgtataac gagctgacca aagtgaaata
     2521 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa aggccatcgt
     2581 ggacctgctg ttcaagacca accggaaagt gaccgtgaag cagctgaaag aggactactt
     2641 caagaaaatc gagtgcttcg actccgtgga aatctccggc gtggaagatc ggttcaacgc
     2701 ctccctgggc acataccacg atctgctgaa aattatcaag gacaaggact tcctggacaa
     2761 tgaggaaaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggacag
     2821 agagatgatc gaggaacggc tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa
     2881 gcagctgaag cggcggagat acaccggctg gggcaggctg agccggaagc tgatcaacgg
     2941 catccgggac aagcagtccg gcaagacaat cctggatttc ctgaagtccg acggcttcgc
     3001 caacagaaac ttcatgcagc tgatccacga cgacagcctg acctttaaag aggacatcca
     3061 gaaagcccag gtgtccggcc agggcgatag cctgcacgag cacattgcca atctggccgg
     3121 cagccccgcc attaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
     3181 agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa atggccagag agaaccagac
     3241 cacccagaag ggacagaaga acagccgcga gagaatgaag cggatcgaag agggcatcaa
     3301 agagctgggc agccagatcc tgaaagaaca ccccgtggaa aacacccagc tgcagaacga
     3361 gaagctgtac ctgtactacc tgcagaatgg gcgggatatg tacgtggacc aggaactgga
     3421 catcaaccgg ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga
     3481 cgactccatc gacaacaagg tgctgaccag aagcgacaag aaccggggca agagcgacaa
     3541 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcggcagc tgctgaacgc
     3601 caagctgatt acccagagaa agttcgacaa tctgaccaag gccgagagag gcggcctgag
     3661 cgaactggat aaggccggct tcatcaagag acagctggtg gaaacccggc agatcacaaa
     3721 gcacgtggca cagatcctgg actcccggat gaacactaag tacgacgaga atgacaagct
     3781 gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga
     3841 tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
     3901 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
     3961 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg agcaggaaat
     4021 cggcaaggct accgccaagt acttcttcta cagcaacatc atgaactttt tcaagaccga
     4081 gattaccctg gccaacggcg agatccggaa gcggcctctg atcgagacaa acggcgaaac
     4141 cggggagatc gtgtgggata agggccggga ttttgccacc gtgcggaaag tgctgagcat
     4201 gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
     4261 tatcctgccc aagaggaaca gcgataagct gatcgccaga aagaaggact gggaccctaa
     4321 gaagtacggc ggcttcgaca gccccaccgt ggcctattct gtgctggtgg tggccaaagt
     4381 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag ctgctgggga tcaccatcat
     4441 ggaaagaagc agcttcgaga agaatcccat cgactttctg gaagccaagg gctacaaaga
     4501 agtgaaaaag gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg
     4561 ccggaagaga atgctggcct ctgccggcga actgcagaag ggaaacgaac tggccctgcc
     4621 ctccaaatat gtgaacttcc tgtacctggc cagccactat gagaagctga agggctcccc
     4681 cgaggataat gagcagaaac agctgtttgt ggaacagcac aagcactacc tggacgagat
     4741 catcgagcag atcagcgagt tctccaagag agtgatcctg gccgacgcta atctggacaa
     4801 agtgctgtcc gcctacaaca agcaccggga taagcccatc agagagcagg ccgagaatat
     4861 catccacctg tttaccctga ccaatctggg agcccctgcc gccttcaagt actttgacac
     4921 caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
     4981 ccagagcatc accggcctgt acgagacacg gatcgacctg tctcagctgg gaggcgacaa
     5041 aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaagggcg gcggatctgg
     5101 tggtgggtct ggcggaggct cgaaggaatt ctatattagt atcgagacag tgggaaacaa
     5161 cattgtagag cggtatatcg atgaaaacgg caaagaaagg actcgagagg tcgaatacct
     5221 tccgaccatg tttcgccact gtaaagaaga atctaagtac aaagatattt acgggaagaa
     5281 ttgcgctccc caaaaatttc cctccatgaa ggatgctcga gactggatga agcgcatgga
     5341 ggacataggt ctcgaagcat tggggatgaa cgattttaag ttggcttaca tctccgacac
     5401 ttacgggtca gaaatagtgt acgataggaa gttcgttcgc gtggcaaatt gcgatataga
     5461 ggtcactgga gataagttcc cggacccgat gaaggcggag tacgaaattg acgctataac
     5521 acactatgac tcaatcgacg accggttcta tgtatttgac ctgctcaatt ccatgtacgg
     5581 gtctgtaagc aagtgggacg ctaaactcgc ggctaaactt gactgtgaag gaggggatga
     5641 agtacctcag gaaatcttgg acagggtaat ctacatgccc tttgacaatg aacgagatat
     5701 gcttatggag tacattaatt tgtgggagca aaagcgcccc gcaatattta caggctggaa
     5761 catagaaggg ttcgatgtac cgtatattat gaatcgggta aagatgatcc tcggagagag
     5821 aagcatgaaa agattttcac ctattggcag agtgaaatct aagttgatac aaaacatgta
     5881 tggctcaaaa gagatctatt caatagatgg agttagcata ctcgactacc tggatctgta
     5941 taaaaagttt gcttttacca acttgcctag cttctccctt gaaagtgtcg cccaacacga
     6001 gaccaagaaa ggtaagctgc cgtacgatgg cccgattaat aaactgcgcg agaccaacca
     6061 tcaaagatat attagctaca acataattga tgtcgaatct gtgcaagcca ttgataagat
     6121 aaggggcttt atcgaccttg tcctgtcaat gtcctattac gccaaaatgc cgttctcagg
     6181 tgtaatgtca cccataaaga cgtgggatgc gatcatcttc aattctctca agggagagca
     6241 caaggtgatc ccccaacagg ggtcccacgt aaagcagtcc ttcccgggag cttttgtctt
     6301 tgagccaaag ccgatcgccc gaaggtatat catgtctttc gaccttacgt cactttaccc
     6361 ttcaattatt cgacaagtga atatatcacc cgagactatc cggggccagt ttaaggtaca
     6421 cccaatccat gagtacatag ccggtacagc cccaaaaccc agtgacgaat actcttgcag
     6481 ccctaatggg tggatgtacg acaaacacca ggagggcata atcccaaagg aaatcgcgaa
     6541 agtatttttc caacggaaag actggaagaa aaagatgttc gcggaggaaa tgaacgccga
     6601 ggctattaaa aaaatcatta tgaagggagc gggtagctgt tctaccaagc cagaggtaga
     6661 gcgctacgtc aaattcagtg atgacttcct taatgagctg agtaactaca cagagtctgt
     6721 actgaactca ctgattgagg aatgtgaaaa agccgcaaca cttgctaata ccaatcaact
     6781 gaatcggaag atcctgatta attcactgta tggcgccttg ggcaacattc atttcagata
     6841 ctacgacctc aggaatgcca cggccattac aattttcggt caggtcggga tccagtggat
     6901 cgcccgaaaa atcaacgagt acctcaataa agtgtgtggt accaatgacg aggattttat
     6961 cgcagcaggc gataccgata gcgtgtatgt ttgcgtcgac aaggtcattg aaaaggtagg
     7021 gctggatcgg tttaaggagc agaatgatct tgtcgagttt atgaaccagt ttggtaaaaa
     7081 aaagatggaa ccgatgatag atgtagcgta ccgagaactt tgtgactaca tgaataatcg
     7141 cgagcacttg atgcacatgg acagggaagc gatttcatgc cccccactcg gttcaaaggg
     7201 cgtagggggt ttctggaaag ctaagaaacg gtacgccctc aacgtctatg acatggaaga
     7261 caagaggttc gcggaacctc atttgaagat aatggggatg gagacgcaac agtcctcaac
     7321 tccaaaggct gtgcaagagg ctctggaaga aagcatacga cgcatactcc aggaggggga
     7381 agagagtgtt caggagtatt ataaaaactt tgaaaaggag taccgccagc ttgactacaa
     7441 ggtaatcgcg gaggttaaga ctgcgaatga tatcgccaaa tatgatgata aaggatggcc
     7501 cggtttcaaa tgccctttcc atatacgagg ggtcctcacc taccgccgcg ccgtgtctgg
     7561 tctgggggtc gcaccaattc tcgacggaaa taaggttatg gtactcccac tccgcgaggg
     7621 gaatccgttt ggtgacaaat gcatcgcctg gccgtctggt acggagctcc ccaaggaaat
     7681 acgcagcgac gtcctcagtt ggatcgacca ctccacactg tttcagaagt cattcgttaa
     7741 acctctggcc gggatgtgtg aatccgcggg tatggactac gaggagaaag cttcattgga
     7801 ctttcttttc gggggtggtt ctccaaagaa gaagcggaag gtctagccta gagctcgctg
     7861 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     7921 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     7981 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     8041 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     8101 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     8161 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     8221 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     8281 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     8341 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     8401 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     8461 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     8521 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     8581 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     8641 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     8701 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     8761 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     8821 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     8881 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     8941 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     9001 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     9061 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     9121 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     9181 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     9241 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     9301 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     9361 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     9421 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     9481 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     9541 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     9601 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     9661 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     9721 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     9781 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     9841 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     9901 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     9961 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
    10021 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
    10081 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
    10141 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
    10201 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
    10261 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
    10321 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
    10381 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
    10441 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
    10501 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
    10561 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
    10621 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
    10681 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
    10741 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
    10801 ttttgctggc cttttgctca catgtcagac aaatggctct agagaccggc gccgctacag
    10861 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
    10921 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
    10981 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
    11041 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc
    11101 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
    11161 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
    11221 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
    11281 ccaccggtgg tctcttctag
//
