LOCUS       sequence-123056-       10801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for sgRNA and for Expression of Cas9 linked via 
            T2A to mCherry and to Ad4 E1B55K via P2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10801)
  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 10801)
  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-mcherry-P2A-Ad4E1B
FEATURES             Location/Qualifiers
     source          1..10801
                     /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             5589..6296
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5734..5752)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             6303..6359
                     /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             6366..7814
                     /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(7842..7859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7848..8055
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8064..8204
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8064..8193
                     /label=AAV2 ITR
     rep_origin      8279..8734
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8366..8385)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8576..8597
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8751..8770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8870..8892
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8930..8948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9016..9120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9121..9189,9190..9981)
                     /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(9339..9358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10152..10740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10641..10660
                     /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 ggcgaggagg ataacatggc catcatcaag gagttcatgc
     5641 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     5701 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     5761 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     5821 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     5881 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     5941 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctcagacg
     6001 gccccgtaat gcagaagaaa accatgggct gggaggcctc ctccgagcgg atgtaccccg
     6061 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     6121 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     6181 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     6241 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagctcg
     6301 aggcaacaaa cttctcacta ctcaaacaag caggtgacgt ggaggagaat cccgggcctc
     6361 ttaagatgga gagccgaaat ccttttcagc agggactgcc agccgggttc ctgtcttcat
     6421 cctttgtgga gaatatggag gtgccagccc ccgagtgcaa cctgcgactg ctggcaggaa
     6481 ccgcagctcg ccacagtgag gatcctgaat caccagcagc cggcggaagt aggagagaga
     6541 gtgaatcacg accaggacct tcaggaggag gagtcgctga cctgttccca gaactgcatc
     6601 gcgtgctgac acgaagctcc tctggccgag agaggggaat caagagagag cggcacgatg
     6661 aaaccaatca taggattgag ctgacagtgg ggctgatgtc tcgcaaacga cctgagactg
     6721 tctggtggta cgaagtgcag agcaccggca cagacgaagt gtccgtcatg cacgagaggt
     6781 tttctctgga acaggtgaag acctgttggc tggagcctga agacgattgg gaggtggcca
     6841 tcagaaacta cgccaagctg gctctgagac cagaccggaa gtacaagatc actaagctga
     6901 tcaacatccg gaatgcttgc tatatcagcg gcaatggagc agaggtcgaa atttgtctgc
     6961 aggatagggt ggccttcaga tgctgtatga tgaacatgta ccccggggtg gtcgacatgg
     7021 atggcgtgac tttcatgaat atgaggttta gaggggatgg ctataacggc accgtcttca
     7081 tggctaatac aaagctgact gtgcatggat gctccttctt tgggtttaac aatacctgta
     7141 tcgaggcttg gggacaagtg ggggtcaagg gctgcagctt ctccgcaaac tggatgggag
     7201 tggtcgggag gacaaaatca atgctgagcg tgaagaaatg cctgtttgaa agatgtcacc
     7261 tgggcgtgat gagcgaggga gaagcacgga ttcgccattg tgcctccact gagaccggct
     7321 gcttcgtgct gtgcaaagga aacgcaaaga tcaaacacaa tatgatttgc ggagcctctg
     7381 acgagagagg gtaccagatg ctgacatgtg ccgggggcaa cagtcatatg ctggctgcag
     7441 tgcacgtcgc ttctcatagt cgaaagccat ggcccgagtt cgaacacaac gtgatgacta
     7501 gatgcaatat gcatctgggg gcccggcgcg gcatgtttat gccataccag tgtaacctga
     7561 attatgtgaa agtcctgctg gaacccgacg tgatgtcccg ggtgtctctg acaggcgtgt
     7621 tcgatatgaa tgtggaggtc tggaagatcc tgcgctacga cgaatataaa actcgatgca
     7681 gggcctgcga gtgtggaggg aagcacgctc ggtttcagcc cgtgtgcgtg gacgtgacag
     7741 aggatctgcg ccctgatcat ctggtcctgt cctgcactgg cactgagttc gggtcaagcg
     7801 gggaagaaag cgactaggaa ttcctagagc tcgctgatca gcctcgactg tgccttctag
     7861 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     7921 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     7981 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag
     8041 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     8101 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     8161 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     8221 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     8281 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     8341 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     8401 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     8461 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     8521 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     8581 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     8641 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     8701 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     8761 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     8821 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     8881 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     8941 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     9001 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     9061 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     9121 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     9181 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     9241 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     9301 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     9361 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     9421 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     9481 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     9541 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     9601 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     9661 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     9721 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     9781 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc
     9841 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     9901 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     9961 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
    10021 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
    10081 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
    10141 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
    10201 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
    10261 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
    10321 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
    10381 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
    10441 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
    10501 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
    10561 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
    10621 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
    10681 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
    10741 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
    10801 t
//
