LOCUS       sequence-449885-        8966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 nuclease and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8966)
  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 8966)
  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: "Sci Rep."; 
            date: "2014-06-23"; volume: "4:5400. doi"; pages: " 
            10.1038/srep05400"
COMMENT     Sequence Label: pX330A-1x2
FEATURES             Location/Qualifiers
     source          1..8966
                     /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..575
                     /label=chicken beta-actin promoter
     intron          576..803
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      815..824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             824..889
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             896..916
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             941..5041
                     /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             941..5041
                     /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             5042..5089
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5117..5134)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5123..5330
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5339..5479
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5339..5468
                     /label=AAV2 ITR
     rep_origin      5554..6009
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5641..5660)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5851..5872
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6026..6045)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6145..6167
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6205..6223)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6291..6395
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6396..6464,6465..7256)
                     /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(6614..6633)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7427..8015
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7916..7935
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8077..8325
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8077..8097
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8248..8267
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8344..8419
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(8505..8810)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     8660..8682
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8674..8691
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8675..8691
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8701..8720
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8701..8719
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8767..8787)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8767..8785)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8806..8822)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8806..8822)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8819..8841)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8830..8846
                     /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(join(8854..8860,8861..8878,8879..8884))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8899..8920
                     /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 cctccccacc cccaattttg
      361 tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggggc
      421 gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg
      481 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
      541 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
      601 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
      661 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
      721 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
      781 tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata aggaccacga
      841 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
      901 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
      961 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     1021 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     1081 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1141 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     1201 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     1261 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     1321 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     1381 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     1441 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     1501 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     1561 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     1621 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     1681 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     1741 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     1801 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     1861 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     1921 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     1981 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     2041 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     2101 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     2161 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     2221 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     2281 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     2341 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     2401 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     2461 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     2521 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     2581 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     2641 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     2701 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     2761 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     2821 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     2881 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     2941 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     3001 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     3061 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     3121 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     3181 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     3241 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     3301 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     3361 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     3421 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     3481 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     3541 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     3601 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     3661 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     3721 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     3781 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     3841 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     3901 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     3961 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     4021 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     4081 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     4141 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     4201 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     4261 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     4321 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     4381 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     4441 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     4501 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     4561 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     4621 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     4681 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     4741 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     4801 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     4861 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     4921 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     4981 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     5041 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct
     5101 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5161 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5221 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5281 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag
     5341 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     5401 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     5461 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5521 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     5581 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     5641 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     5701 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     5761 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     5821 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     5881 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     5941 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     6001 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     6061 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     6121 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     6181 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     6241 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6301 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6361 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6421 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6481 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6541 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6601 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6661 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6721 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6781 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6841 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6901 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6961 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     7021 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     7081 ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc
     7141 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     7201 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     7261 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     7321 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     7381 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7441 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7501 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7561 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7621 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7681 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7741 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7801 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7861 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7921 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     7981 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     8041 ggttcctggc cttttgctgg ccttttgctc acatgtgagg gcctatttcc catgattcct
     8101 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     8161 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     8221 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     8281 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgggtc ttcgagaaga
     8341 cctgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     8401 agtggcaccg agtcggtgct tttttgtttt agagctagaa atagcaagtt aaaataaggc
     8461 tagtccgttt ttagcgcgtg cgccaattct gcagacaaat ggctctagag accggcgccg
     8521 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     8581 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     8641 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg
     8701 taatacgact cactataggg cgaattgggt accgggcccc ccctcgaggt cctccagctt
     8761 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     8821 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     8881 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     8941 cgctttccac cggtggtctc ttctag
//
