LOCUS       sequence-107960-       10813 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for a sgRNA against the mouse Rosa26 locus and 
            Cas9 linked via T2A to BFP linked to the Ad4 E1B55K gene via P2A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10813)
  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 10813)
  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-sgRosa26-1_CBh-Cas9-T2A-BFP-P2A-Ad4E1B
FEATURES             Location/Qualifiers
     source          1..10813
                     /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        277..352
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        449..734
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        736..1013
                     /label=chicken beta-actin promoter
     intron          1014..1242
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1254..1263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1263..1328
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1335..1355
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1380..5480
                     /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             1380..5480
                     /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             5481..5528
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5532..5585
                     /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(5592..5594,5595..5597,5598..6308)
                     /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             6315..6371
                     /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             6378..7826
                     /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(7854..7871)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7860..8067
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   8076..8216
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   8076..8205
                     /label=AAV2 ITR
     rep_origin      8291..8746
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8378..8397)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8588..8609
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8763..8782)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8882..8904
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8942..8960)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9028..9132
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9133..9201,9202..9993)
                     /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(9351..9370)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10164..10752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10653..10672
                     /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 acaccgactc cagtctttct agaagagttt tagagctaga aatagcaagt
      301 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt
      361 tttagagcta gaaatagcaa gttaaaataa ggctagtccg tttttagcgc gtgcgccaat
      421 tctgcagaca aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg
      481 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
      541 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      601 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      661 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      721 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
      781 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
      841 cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
      901 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
      961 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
     1021 ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
     1081 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
     1141 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
     1201 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca
     1261 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
     1321 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
     1381 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     1441 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     1501 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     1561 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     1621 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     1681 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     1741 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     1801 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     1861 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     1921 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     1981 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     2041 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     2101 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     2161 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     2221 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     2281 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     2341 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     2401 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     2461 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     2521 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     2581 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     2641 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     2701 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     2761 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     2821 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     2881 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     2941 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     3001 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     3061 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     3121 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     3181 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     3241 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     3301 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     3361 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     3421 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     3481 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     3541 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     3601 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     3661 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     3721 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     3781 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     3841 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
     3901 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
     3961 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     4021 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
     4081 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     4141 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     4201 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     4261 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     4321 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     4381 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     4441 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     4501 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     4561 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     4621 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     4681 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
     4741 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     4801 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     4861 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     4921 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     4981 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     5041 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     5101 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     5161 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     5221 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
     5281 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     5341 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     5401 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     5461 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     5521 agaaaaagct tgagggcaga ggaagtctgc taacatgcgg tgacgtggag gagaatcccg
     5581 gccctgctag catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     5641 tcgagctgga cggcgacgta aacggccaca agttcagcgt gaggggcgag ggcgagggcg
     5701 atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     5761 cctggcccac cctcgtgacc accctgagcc acggcgtgca gtgcttcgcc cgctaccccg
     5821 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     5881 gcaccatctt cttcaaggac gacggcacct acaagacccg cgccgaggtg aagttcgagg
     5941 gcgacaccct ggtgaaccgc atcgagctga agggcgtcga cttcaaggag gacggcaaca
     6001 tcctggggca caagctggag tacaacttca acagccacaa catctatatc atggccgtca
     6061 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacgtggag gacggcagcg
     6121 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     6181 ccgacagcca ctacctgagc acccagtccg tgctgagcaa agaccccaac gagaagcgcg
     6241 atcacatggt cctgctggag ttccgcaccg ccgccgggat cactctcggc atggacgagc
     6301 tgtacaagct cgaggcaaca aacttctcac tactcaaaca agcaggtgac gtggaggaga
     6361 atcccgggcc tcttaagatg gagagccgaa atccttttca gcagggactg ccagccgggt
     6421 tcctgtcttc atcctttgtg gagaatatgg aggtgccagc ccccgagtgc aacctgcgac
     6481 tgctggcagg aaccgcagct cgccacagtg aggatcctga atcaccagca gccggcggaa
     6541 gtaggagaga gagtgaatca cgaccaggac cttcaggagg aggagtcgct gacctgttcc
     6601 cagaactgca tcgcgtgctg acacgaagct cctctggccg agagagggga atcaagagag
     6661 agcggcacga tgaaaccaat cataggattg agctgacagt ggggctgatg tctcgcaaac
     6721 gacctgagac tgtctggtgg tacgaagtgc agagcaccgg cacagacgaa gtgtccgtca
     6781 tgcacgagag gttttctctg gaacaggtga agacctgttg gctggagcct gaagacgatt
     6841 gggaggtggc catcagaaac tacgccaagc tggctctgag accagaccgg aagtacaaga
     6901 tcactaagct gatcaacatc cggaatgctt gctatatcag cggcaatgga gcagaggtcg
     6961 aaatttgtct gcaggatagg gtggccttca gatgctgtat gatgaacatg taccccgggg
     7021 tggtcgacat ggatggcgtg actttcatga atatgaggtt tagaggggat ggctataacg
     7081 gcaccgtctt catggctaat acaaagctga ctgtgcatgg atgctccttc tttgggttta
     7141 acaatacctg tatcgaggct tggggacaag tgggggtcaa gggctgcagc ttctccgcaa
     7201 actggatggg agtggtcggg aggacaaaat caatgctgag cgtgaagaaa tgcctgtttg
     7261 aaagatgtca cctgggcgtg atgagcgagg gagaagcacg gattcgccat tgtgcctcca
     7321 ctgagaccgg ctgcttcgtg ctgtgcaaag gaaacgcaaa gatcaaacac aatatgattt
     7381 gcggagcctc tgacgagaga gggtaccaga tgctgacatg tgccgggggc aacagtcata
     7441 tgctggctgc agtgcacgtc gcttctcata gtcgaaagcc atggcccgag ttcgaacaca
     7501 acgtgatgac tagatgcaat atgcatctgg gggcccggcg cggcatgttt atgccatacc
     7561 agtgtaacct gaattatgtg aaagtcctgc tggaacccga cgtgatgtcc cgggtgtctc
     7621 tgacaggcgt gttcgatatg aatgtggagg tctggaagat cctgcgctac gacgaatata
     7681 aaactcgatg cagggcctgc gagtgtggag ggaagcacgc tcggtttcag cccgtgtgcg
     7741 tggacgtgac agaggatctg cgccctgatc atctggtcct gtcctgcact ggcactgagt
     7801 tcgggtcaag cggggaagaa agcgactagg aattcctaga gctcgctgat cagcctcgac
     7861 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     7921 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     7981 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     8041 ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc
     8101 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     8161 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     8221 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     8281 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     8341 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     8401 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     8461 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     8521 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     8581 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     8641 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     8701 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     8761 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     8821 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     8881 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     8941 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     9001 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     9061 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     9121 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     9181 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     9241 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     9301 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     9361 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     9421 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     9481 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     9541 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     9601 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     9661 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     9721 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     9781 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     9841 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     9901 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     9961 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
    10021 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
    10081 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
    10141 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
    10201 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
    10261 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
    10321 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
    10381 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
    10441 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
    10501 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
    10561 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
    10621 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
    10681 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
    10741 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
    10801 tttgctcaca tgt
//
