LOCUS       sequence-193778-        9026 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression vector for sgRNAs cloned into the BbsI sites and for 
            expression of Cas9 linked to mCherry via a T2A peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 9026)
  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 9026)
  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
FEATURES             Location/Qualifiers
     source          1..9026
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      227..236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             236..301
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             308..328
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             353..4453
                     /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             353..4453
                     /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             4454..4501
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4505..4558
                     /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             4565..5275
                     /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(4710..4728)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5300..5317)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5306..5513
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5522..5662
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5522..5651
                     /label=AAV2 ITR
     rep_origin      5737..6192
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5824..5843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6034..6055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6209..6228)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6328..6350
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6388..6406)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6474..6578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6579..6647,6648..7439)
                     /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(6797..6816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7610..8198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8099..8118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8260..8508
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8260..8280
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8431..8450
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8527..8602
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8699..8984
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
       61 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
      121 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
      181 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
      241 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
      301 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
      361 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
      421 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
      481 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
      541 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
      601 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
      661 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
      721 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
      781 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
      841 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
      901 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
      961 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1021 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     1081 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     1141 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     1201 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     1261 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     1321 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     1381 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     1441 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     1501 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     1561 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     1621 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     1681 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     1741 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     1801 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     1861 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     1921 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     1981 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     2041 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     2101 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     2161 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     2221 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     2281 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     2341 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     2401 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     2461 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     2521 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     2581 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     2641 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     2701 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     2761 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     2821 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     2881 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     2941 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3001 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     3061 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     3121 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     3181 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     3241 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     3301 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     3361 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     3421 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     3481 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     3541 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     3601 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     3661 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     3721 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     3781 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     3841 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     3901 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     3961 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4021 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     4081 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     4141 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     4201 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     4261 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     4321 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     4381 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     4441 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     4501 gcttgagggc agaggaagtc tgctaacatg cggtgacgtg gaggagaatc ccggccctgc
     4561 tagcatggtg agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt
     4621 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     4681 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     4741 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     4801 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     4861 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     4921 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct cagacggccc
     4981 cgtaatgcag aagaaaacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     5041 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     5101 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     5161 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     5221 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagaatt
     5281 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5341 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     5401 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     5461 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     5521 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5581 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5641 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5701 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5761 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5821 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5881 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5941 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     6001 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     6061 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     6121 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6181 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6241 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6301 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6361 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6421 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6481 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6541 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6601 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6661 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6721 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6781 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6841 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6901 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6961 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     7021 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7081 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7141 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7201 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7261 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7321 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7381 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7441 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7501 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7561 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7621 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7681 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7741 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7801 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7861 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7921 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7981 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     8041 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8101 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8161 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8221 tacggttcct ggccttttgc tggccttttg ctcacatgtg agggcctatt tcccatgatt
     8281 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     8341 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     8401 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     8461 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg gtcttcgaga
     8521 agacctgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
     8581 aaaagtggca ccgagtcggt gcttttttgt tttagagcta gaaatagcaa gttaaaataa
     8641 ggctagtccg tttttagcgc gtgcgccaat tctgcagaca aatggctcta gaggtacccg
     8701 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     8761 cgtcaatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     8821 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     8881 tcaatgacgg taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc
     8941 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     9001 cgttctgctt cactctcccc atctcc
//
