LOCUS       sequence-107961-       10810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for sgRNA and for Expression of Cas9 linked to BFP
            via T2A linked to Ad4 E1B via P2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10810)
  AUTHORS   Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R
  TITLE     Increasing the efficiency of homology-directed repair for 
            CRISPR-Cas9-induced precise gene editing in mammalian cells.
  JOURNAL   Nat Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198.
  PUBMED    25803306
REFERENCE   2  (bases 1 to 10810)
  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: "Nat 
            Biotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198."
COMMENT     Sequence Label: pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E1B
FEATURES             Location/Qualifiers
     source          1..10810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..255
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7..27
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     178..197
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        274..349
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        446..731
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        733..1010
                     /label=chicken beta-actin promoter
     intron          1011..1239
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1251..1260
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1260..1325
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1332..1352
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1377..5477
                     /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             1377..5477
                     /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             5478..5525
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5529..5582
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5589..5591,5592..5594,5595..6305)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             6312..6368
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             6375..7823
                     /codon_start=1
                     /product="adenovirus 4 E1B 55K protein, which mediates the 
                     ubiquitination and proteasomal degradation of DNA ligase IV
                     (Baker et al., 2007)"
                     /label=E1B
                     /translation="MESRNPFQQGLPAGFLSSSFVENMEVPAPECNLRLLAGTAARHSE
                     DPESPAAGGSRRESESRPGPSGGGVADLFPELHRVLTRSSSGRERGIKRERHDETNHRI
                     ELTVGLMSRKRPETVWWYEVQSTGTDEVSVMHERFSLEQVKTCWLEPEDDWEVAIRNYA
                     KLALRPDRKYKITKLINIRNACYISGNGAEVEICLQDRVAFRCCMMNMYPGVVDMDGVT
                     FMNMRFRGDGYNGTVFMANTKLTVHGCSFFGFNNTCIEAWGQVGVKGCSFSANWMGVVG
                     RTKSMLSVKKCLFERCHLGVMSEGEARIRHCASTETGCFVLCKGNAKIKHNMICGASDE
                     RGYQMLTCAGGNSHMLAAVHVASHSRKPWPEFEHNVMTRCNMHLGARRGMFMPYQCNLN
                     YVKVLLEPDVMSRVSLTGVFDMNVEVWKILRYDEYKTRCRACECGGKHARFQPVCVDVT
                     EDLRPDHLVLSCTGTEFGSSGEESD"
     primer_bind     complement(7851..7868)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7857..8064
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8073..8213
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8073..8202
                     /label=AAV2 ITR
     rep_origin      8288..8743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8375..8394)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8585..8606
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8760..8779)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8879..8901
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8939..8957)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9025..9129
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9130..9198,9199..9990)
                     /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(9348..9367)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10161..10749
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10650..10669
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccccacgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
       61 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      121 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      181 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      241 aaaggacgaa acaccgggtc ttcgagaaga cctgttttag agctagaaat agcaagttaa
      301 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt
      361 agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct
      421 gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct
      481 gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt
      541 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      601 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg
      661 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      721 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
      781 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
      841 gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga
      901 ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
      961 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
     1021 cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
     1081 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
     1141 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
     1201 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg
     1261 actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg
     1321 ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca
     1381 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
     1441 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
     1501 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
     1561 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
     1621 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
     1681 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
     1741 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
     1801 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
     1861 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     1921 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     1981 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     2041 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     2101 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     2161 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     2221 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     2281 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     2341 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     2401 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     2461 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     2521 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     2581 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     2641 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     2701 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     2761 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     2821 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     2881 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     2941 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     3001 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     3061 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     3121 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     3181 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     3241 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     3301 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     3361 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     3421 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     3481 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     3541 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     3601 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     3661 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     3721 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     3781 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     3841 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     3901 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     3961 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     4021 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     4081 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     4141 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     4201 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     4261 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     4321 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     4381 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     4441 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     4501 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     4561 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     4621 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     4681 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     4741 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     4801 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     4861 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     4921 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     4981 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     5041 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     5101 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     5161 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     5221 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     5281 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     5341 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     5401 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     5461 ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga
     5521 aaaagcttga gggcagagga agtctgctaa catgcggtga cgtggaggag aatcccggcc
     5581 ctgctagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     5641 agctggacgg cgacgtaaac ggccacaagt tcagcgtgag gggcgagggc gagggcgatg
     5701 ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     5761 ggcccaccct cgtgaccacc ctgagccacg gcgtgcagtg cttcgcccgc taccccgacc
     5821 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     5881 ccatcttctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg
     5941 acaccctggt gaaccgcatc gagctgaagg gcgtcgactt caaggaggac ggcaacatcc
     6001 tggggcacaa gctggagtac aacttcaaca gccacaacat ctatatcatg gccgtcaagc
     6061 agaagaacgg catcaaggtg aacttcaaga tccgccacaa cgtggaggac ggcagcgtgc
     6121 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     6181 acagccacta cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag aagcgcgatc
     6241 acatggtcct gctggagttc cgcaccgccg ccgggatcac tctcggcatg gacgagctgt
     6301 acaagctcga ggcaacaaac ttctcactac tcaaacaagc aggtgacgtg gaggagaatc
     6361 ccgggcctct taagatggag agccgaaatc cttttcagca gggactgcca gccgggttcc
     6421 tgtcttcatc ctttgtggag aatatggagg tgccagcccc cgagtgcaac ctgcgactgc
     6481 tggcaggaac cgcagctcgc cacagtgagg atcctgaatc accagcagcc ggcggaagta
     6541 ggagagagag tgaatcacga ccaggacctt caggaggagg agtcgctgac ctgttcccag
     6601 aactgcatcg cgtgctgaca cgaagctcct ctggccgaga gaggggaatc aagagagagc
     6661 ggcacgatga aaccaatcat aggattgagc tgacagtggg gctgatgtct cgcaaacgac
     6721 ctgagactgt ctggtggtac gaagtgcaga gcaccggcac agacgaagtg tccgtcatgc
     6781 acgagaggtt ttctctggaa caggtgaaga cctgttggct ggagcctgaa gacgattggg
     6841 aggtggccat cagaaactac gccaagctgg ctctgagacc agaccggaag tacaagatca
     6901 ctaagctgat caacatccgg aatgcttgct atatcagcgg caatggagca gaggtcgaaa
     6961 tttgtctgca ggatagggtg gccttcagat gctgtatgat gaacatgtac cccggggtgg
     7021 tcgacatgga tggcgtgact ttcatgaata tgaggtttag aggggatggc tataacggca
     7081 ccgtcttcat ggctaataca aagctgactg tgcatggatg ctccttcttt gggtttaaca
     7141 atacctgtat cgaggcttgg ggacaagtgg gggtcaaggg ctgcagcttc tccgcaaact
     7201 ggatgggagt ggtcgggagg acaaaatcaa tgctgagcgt gaagaaatgc ctgtttgaaa
     7261 gatgtcacct gggcgtgatg agcgagggag aagcacggat tcgccattgt gcctccactg
     7321 agaccggctg cttcgtgctg tgcaaaggaa acgcaaagat caaacacaat atgatttgcg
     7381 gagcctctga cgagagaggg taccagatgc tgacatgtgc cgggggcaac agtcatatgc
     7441 tggctgcagt gcacgtcgct tctcatagtc gaaagccatg gcccgagttc gaacacaacg
     7501 tgatgactag atgcaatatg catctggggg cccggcgcgg catgtttatg ccataccagt
     7561 gtaacctgaa ttatgtgaaa gtcctgctgg aacccgacgt gatgtcccgg gtgtctctga
     7621 caggcgtgtt cgatatgaat gtggaggtct ggaagatcct gcgctacgac gaatataaaa
     7681 ctcgatgcag ggcctgcgag tgtggaggga agcacgctcg gtttcagccc gtgtgcgtgg
     7741 acgtgacaga ggatctgcgc cctgatcatc tggtcctgtc ctgcactggc actgagttcg
     7801 ggtcaagcgg ggaagaaagc gactaggaat tcctagagct cgctgatcag cctcgactgt
     7861 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     7921 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     7981 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     8041 agagaatagc aggcatgctg gggagcggcc gcaggaaccc ctagtgatgg agttggccac
     8101 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     8161 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     8221 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     8281 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     8341 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     8401 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     8461 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     8521 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     8581 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt
     8641 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     8701 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     8761 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     8821 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     8881 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     8941 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     9001 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     9061 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     9121 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     9181 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     9241 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     9301 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     9361 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     9421 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     9481 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     9541 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     9601 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     9661 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     9721 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     9781 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     9841 cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     9901 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     9961 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
    10021 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
    10081 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
    10141 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
    10201 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
    10261 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
    10321 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
    10381 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
    10441 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
    10501 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
    10561 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
    10621 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
    10681 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
    10741 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
    10801 gctcacatgt
//
