LOCUS       sequence-91083-e        8963 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9 nickase and gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8963)
  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 8963)
  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_D10A-1x3
FEATURES             Location/Qualifiers
     source          1..8963
                     /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..801
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      813..822
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             822..887
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             894..914
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(939..962,963..965,966..5039)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             939..5039
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5040..5087
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5115..5132)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5121..5328
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5337..5477
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5337..5466
                     /label=AAV2 ITR
     rep_origin      5552..6007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5639..5658)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5849..5870
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6024..6043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6143..6165
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6203..6221)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6289..6393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6394..6462,6463..7254)
                     /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(6612..6631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7425..8013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7914..7933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8075..8323
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8075..8095
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8246..8265
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8342..8417
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(8503..8808)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGL"
     primer_bind     8658..8680
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8672..8689
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8673..8689
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8699..8718
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        8699..8717
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8765..8785)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(8765..8783)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8804..8820)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8804..8820)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8817..8839)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8828..8844
                     /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(8852..8858,8859..8876,8877..8882))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8897..8918
                     /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 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg
      481 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      541 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc
      601 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
      661 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
      721 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
      781 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg
      841 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga
      901 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc
      961 tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     1021 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     1081 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
     1141 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     1201 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     1261 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     1321 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     1381 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     1441 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     1501 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     1561 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     1621 tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga
     1681 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     1741 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     1801 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     1861 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     1921 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     1981 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
     2041 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     2101 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     2161 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     2221 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     2281 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     2341 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
     2401 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     2461 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
     2521 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg
     2581 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     2641 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     2701 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     2761 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     2821 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     2881 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     2941 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     3001 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     3061 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     3121 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     3181 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     3241 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     3301 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     3361 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     3421 acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg
     3481 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca
     3541 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg
     3601 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     3661 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     3721 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc
     3781 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     3841 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
     3901 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
     3961 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
     4021 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
     4081 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
     4141 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca
     4201 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
     4261 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta
     4321 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
     4381 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
     4441 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
     4501 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
     4561 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
     4621 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
     4681 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga
     4741 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
     4801 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata
     4861 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
     4921 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
     4981 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca
     5041 aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag
     5101 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5161 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5221 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5281 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     5341 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     5401 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     5461 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5521 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     5581 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5641 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5701 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5761 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5821 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5881 aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5941 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     6001 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     6061 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6121 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6181 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     6241 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     6301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6481 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6541 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6781 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7081 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7261 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7321 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7501 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7561 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7621 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7681 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7741 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7801 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7861 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7921 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7981 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     8041 ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc
     8101 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     8161 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     8221 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     8281 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc
     8341 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     8401 tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta
     8461 gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagagac cggcgccgct
     8521 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     8581 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     8641 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
     8701 atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcc tccagctttt
     8761 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     8821 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     8881 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     8941 ctttccaccg gtggtctctc tag
//
