LOCUS       sequence-195323-        9697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9n (D10A Nickase mutant) with 2A-EGFP and a cloning backbone 
            for 2 custom gRNAs which can be cloned in via a one-step reaction. 
            For generation of DSBs with no off-target cuts..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9697)
  AUTHORS   Adikusuma F, Pfitzner C, Thomas PQ
  TITLE     Versatile single-step-assembly CRISPR/Cas9 vectors for dual gRNA 
            expression.
  JOURNAL   PLoS One. 2017 Dec 6;12(12):e0187236. doi: 
            10.1371/journal.pone.0187236. eCollection 2017.
  PUBMED    29211736
REFERENCE   2  (bases 1 to 9697)
  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: "PLoS One.";
            date: "2017-12-6"; volume: "12(12):e0187236. doi"; pages: " 
            10.1371/journal.pone.0187236. eCollection 2017"
COMMENT     Sequence Label: pDG461
FEATURES             Location/Qualifiers
     source          1..9697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1325..1345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1370..1393,1394..1396,1397..5470)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1370..5470
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5471..5518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5534..5587
                     /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(5588..5590,5591..6301)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5588..5590,5591..6301)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(5891..5910)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6238..6259
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6329..6346)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6335..6542
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6551..6567
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6551..6567
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_RNA        complement(6610..6685)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(6703..6951)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     repeat_region   6960..7100
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6960..7089
                     /label=AAV2 ITR
     rep_origin      7175..7630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7262..7281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7472..7493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7647..7666)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7766..7788
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7826..7844)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7912..8016
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8017..8085,8086..8877)
                     /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(8235..8254)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9048..9636
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9537..9556
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
     1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     1381 cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag
     2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     2821 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     3421 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     3901 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     4141 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagga
     5521 attcggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc
     5581 tggcccagtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     5641 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     5701 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     5761 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     5821 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     5881 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     5941 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     6001 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     6061 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     6121 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     6181 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     6241 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     6301 ggaattctaa ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     6361 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     6421 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     6481 ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg
     6541 gagcggccgc tctagaacta gtggatcccc cgggctgcag gaattcgata cgaatcctgc
     6601 aggaaaaaag caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt
     6661 aacttgctat ttctagctct aaaacgtctt ctcgaagacc ccggtgtttc gtcctttcca
     6721 caagatatat aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc
     6781 cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca
     6841 cgtattttgt actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac
     6901 agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct cgcggccgca
     6961 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     7021 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     7081 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     7141 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     7201 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     7261 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     7321 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     7381 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     7441 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     7501 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     7561 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     7621 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     7681 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     7741 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     7801 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     7861 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     7921 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     7981 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     8041 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     8101 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     8161 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     8221 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     8281 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     8341 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     8401 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     8461 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     8521 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     8581 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     8641 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     8701 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     8761 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     8821 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     8881 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     8941 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     9001 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     9061 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     9121 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     9181 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     9241 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     9301 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     9361 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     9421 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     9481 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     9541 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     9601 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     9661 cggttcctgg ccttttgctg gccttttgct cacatgt
//
