LOCUS       sequence-83240-e        9654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Csy4 and Cas9 D10A nickase expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9654)
  AUTHORS   Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin
            MJ, Aryee MJ, Joung JK
  TITLE     Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome 
            editing.
  JOURNAL   Nat Biotechnol. 2014 Apr 25. doi: 10.1038/nbt.2908.
  PUBMED    24770325
REFERENCE   2  (bases 1 to 9654)
  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. 2014 Apr 25. doi: 10.1038/nbt.2908."
COMMENT     Sequence Label: pNW3
FEATURES             Location/Qualifiers
     source          1..9654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1763..1772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1769..2329
                     /codon_start=1
                     /product="CRISPR-associated endoribonuclease responsible 
                     for pre-crRNA processing in Pseudomonas aeruginosa 
                     (Haurwitz et al., 2010)"
                     /label=Csy4
                     /translation="MDHYLDIRLRPDPEFPPAQLMSVLFGKLHQALVAQGGDRIGVSFP
                     DLDESRSRLGERLRIHASADDLRALLARPWLEGLRDHLQFGEPAVVPHPTPYRQVSRVQ
                     AKSNPERLRRRLMRRHDLSEEEARKRIPDTVARALDLPFVTLRSQSTGQHFRLFIRHGP
                     LQVTAEEGGFTCYGLSKGGFVPWF"
     CDS             2330..2383
                     /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(2384..5785,5786..5788,5789..6385,6386,6387..6389,
                     6390..6391,6392..6394,6395..6487)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2384..2410,2411..2413,2414..6487)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6494..6514
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6521..6586
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(6676..6695)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6741..6796
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6795..6814)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7157..7173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7157..7173)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7170..7192)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7181..7197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(7205..7211,7212..7229,7230..7235))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7250..7271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7330..7525
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7376..7511
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7531..7665
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7568..7587)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7622..7641
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7733..7750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7904..8492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7984..8003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8663..9454,9455..9523))
                     /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     9286..9305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9524..9628)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc
     1741 gcgggcccgg gatccaccgg tcgccaccat ggaccactac ctcgacattc gcttgcgacc
     1801 ggacccggaa tttcccccgg cgcaactcat gagcgtgctc ttcggcaagc tccaccaggc
     1861 cctggtggca cagggcgggg acaggatcgg cgtgagcttc cccgacctcg acgaaagccg
     1921 ctcccggctg ggcgagcgcc tgcgcattca tgcctcggcg gacgaccttc gtgccctgct
     1981 cgcccggccc tggctggaag ggttgcggga ccatctgcaa ttcggagaac cggcagtcgt
     2041 gcctcacccc acaccgtacc gtcaggtcag tcgggttcag gcgaaaagca atccggaacg
     2101 cctgcggcgg cggctcatgc gccggcacga tctgagtgag gaggaggctc ggaaacgcat
     2161 tcccgatacg gtcgcgagag ccttggacct gcccttcgtc acgctacgca gccagagcac
     2221 cggacagcac ttccgtctct tcatccgcca cgggccgttg caggtgacgg cagaggaagg
     2281 aggattcacc tgttacgggt tgagcaaagg aggtttcgtt ccctggttcg agggcagagg
     2341 aagtctgtta acatgcggtg acgtcgagga gaatcctggc ccaatggata aaaagtattc
     2401 tattggttta gccatcggca ctaattccgt tggatgggct gtcataaccg atgaatacaa
     2461 agtaccttca aagaaattta aggtgttggg gaacacagac cgtcattcga ttaaaaagaa
     2521 tcttatcggt gccctcctat tcgatagtgg cgaaacggca gaggcgactc gcctgaaacg
     2581 aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata tgttacttac aagaaatttt
     2641 tagcaatgag atggccaaag ttgacgattc tttctttcac cgtttggaag agtccttcct
     2701 tgtcgaagag gacaagaaac atgaacggca ccccatcttt ggaaacatag tagatgaggt
     2761 ggcatatcat gaaaagtacc caacgattta tcacctcaga aaaaagctag ttgactcaac
     2821 tgataaagcg gacctgaggt taatctactt ggctcttgcc catatgataa agttccgtgg
     2881 gcactttctc attgagggtg atctaaatcc ggacaactcg gatgtcgaca aactgttcat
     2941 ccagttagta caaacctata atcagttgtt tgaagagaac cctataaatg caagtggcgt
     3001 ggatgcgaag gctattctta gcgcccgcct ctctaaatcc cgacggctag aaaacctgat
     3061 cgcacaatta cccggagaga agaaaaatgg gttgttcggt aaccttatag cgctctcact
     3121 aggcctgaca ccaaatttta agtcgaactt cgacttagct gaagatgcca aattgcagct
     3181 tagtaaggac acgtacgatg acgatctcga caatctactg gcacaaattg gagatcagta
     3241 tgcggactta tttttggctg ccaaaaacct tagcgatgca atcctcctat ctgacatact
     3301 gagagttaat actgagatta ccaaggcgcc gttatccgct tcaatgatca aaaggtacga
     3361 tgaacatcac caagacttga cacttctcaa ggccctagtc cgtcagcaac tgcctgagaa
     3421 atataaggaa atattctttg atcagtcgaa aaacgggtac gcaggttata ttgacggcgg
     3481 agcgagtcaa gaggaattct acaagtttat caaacccata ttagagaaga tggatgggac
     3541 ggaagagttg cttgtaaaac tcaatcgcga agatctactg cgaaagcagc ggactttcga
     3601 caacggtagc attccacatc aaatccactt aggcgaattg catgctatac ttagaaggca
     3661 ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa tcctaacctt
     3721 tcgcatacct tactatgtgg gacccctggc ccgagggaac tctcggttcg catggatgac
     3781 aagaaagtcc gaagaaacga ttactccatg gaattttgag gaagttgtcg ataaaggtgc
     3841 gtcagctcaa tcgttcatcg agaggatgac caactttgac aagaatttac cgaacgaaaa
     3901 agtattgcct aagcacagtt tactttacga gtatttcaca gtgtacaatg aactcacgaa
     3961 agttaagtat gtcactgagg gcatgcgtaa acccgccttt ctaagcggag aacagaagaa
     4021 agcaatagta gatctgttat tcaagaccaa ccgcaaagtg acagttaagc aattgaaaga
     4081 ggactacttt aagaaaattg aatgcttcga ttctgtcgag atctccgggg tagaagatcg
     4141 atttaatgcg tcacttggta cgtatcatga cctcctaaag ataattaaag ataaggactt
     4201 cctggataac gaagagaatg aagatatctt agaagatata gtgttgactc ttaccctctt
     4261 tgaagatcgg gaaatgattg aggaaagact aaaaacatac gctcacctgt tcgacgataa
     4321 ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt cgcggaaact
     4381 tatcaacggg ataagagaca agcaaagtgg taaaactatt ctcgattttc taaagagcga
     4441 cggcttcgcc aataggaact ttatgcagct gatccatgat gactctttaa ccttcaaaga
     4501 ggatatacaa aaggcacagg tttccggaca aggggactca ttgcacgaac atattgcgaa
     4561 tcttgctggt tcgccagcca tcaaaaaggg catactccag acagtcaaag tagtggatga
     4621 gctagttaag gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga tggcacgcga
     4681 aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga gaatagaaga
     4741 gggtattaaa gaactgggca gccagatctt aaaggagcat cctgtggaaa atacccaatt
     4801 gcagaacgag aaactttacc tctattacct acaaaatgga agggacatgt atgttgatca
     4861 ggaactggac ataaaccgtt tatctgatta cgacgtcgat cacattgtac cccaatcctt
     4921 tttgaaggac gattcaatcg acaataaagt gcttacacgc tcggataaga accgagggaa
     4981 aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg aagaactatt ggcggcagct
     5041 cctaaatgcg aaactgataa cgcaaagaaa gttcgataac ttaactaaag ctgagagggg
     5101 tggcttgtct gaacttgaca aggccggatt tattaaacgt cagctcgtgg aaacccgcca
     5161 aatcacaaag catgttgcac agatactaga ttcccgaatg aatacgaaat acgacgagaa
     5221 cgataagctg attcgggaag tcaaagtaat cactttaaag tcaaaattgg tgtcggactt
     5281 cagaaaggat tttcaattct ataaagttag ggagataaat aactaccacc atgcgcacga
     5341 cgcttatctt aatgccgtcg tagggaccgc actcattaag aaatacccga agctagaaag
     5401 tgagtttgtg tatggtgatt acaaagttta tgacgtccgt aagatgatcg cgaaaagcga
     5461 acaggagata ggcaaggcta cagccaaata cttcttttat tctaacatta tgaatttctt
     5521 taagacggaa atcactctgg caaacggaga gatacgcaaa cgacctttaa ttgaaaccaa
     5581 tggggagaca ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg tgagaaaagt
     5641 tttgtccatg ccccaagtca acatagtaaa gaaaactgag gtgcagaccg gagggttttc
     5701 aaaggaatcg attcttccaa aaaggaatag tgataagctc atcgctcgta aaaaggactg
     5761 ggacccgaaa aagtacggtg gcttcgatag ccctacagtt gcctattctg tcctagtagt
     5821 ggcaaaagtt gagaagggaa aatccaagaa actgaagtca gtcaaagaat tattggggat
     5881 aacgattatg gagcgctcgt cttttgaaaa gaaccccatc gacttccttg aggcgaaagg
     5941 ttacaaggaa gtaaaaaagg atctcataat taaactacca aagtatagtc tgtttgagtt
     6001 agaaaatggc cgaaaacgga tgttggctag cgccggagag cttcaaaagg ggaacgaact
     6061 cgcactaccg tctaaatacg tgaatttcct gtatttagcg tcccattacg agaagttgaa
     6121 aggttcacct gaagataacg aacagaagca actttttgtt gagcagcaca aacattatct
     6181 cgacgaaatc atagagcaaa tttcggaatt cagtaagaga gtcatcctag ctgatgccaa
     6241 tctggacaaa gtattaagcg catacaacaa gcacagggat aaacccatac gtgagcaggc
     6301 ggaaaatatt atccatttgt ttactcttac caacctcggc gctccagccg cattcaagta
     6361 ttttgacaca acgatagatc gcaaacgata cacttctacc aaggaggtgc tagacgcgac
     6421 actgattcac caatccatca cgggattata tgaaactcgg atagatttgt cacagcttgg
     6481 gggtgacgga tcccccaaga agaagaggaa agtctcgagc gactacaaag accatgacgg
     6541 tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaagtgaa gcggccgcac
     6601 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     6661 caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct
     6721 tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa
     6781 ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa
     6841 tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg
     6901 ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca
     6961 tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc
     7021 tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg
     7081 actactccca gtcatagctg tccctcttct cttatgaaga tccctcgacc tgcagcccaa
     7141 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     7201 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     7261 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     7321 agcggatccg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     7381 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     7441 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     7501 ttttggaggc ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac
     7561 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     7621 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa
     7681 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     7741 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     7801 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     7861 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     7921 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     7981 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     8041 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat
     8101 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     8161 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     8221 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     8281 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     8341 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     8401 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     8461 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     8521 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     8581 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     8641 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     8701 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     8761 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     8821 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     8881 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     8941 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     9001 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     9061 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     9121 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     9181 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     9241 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     9301 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     9361 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     9421 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     9481 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     9541 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     9601 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
