LOCUS       sequence-447618-        9874 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCAG human codon optimized wild-type SpCas9 expression plasmid with 
            C-terminal NLS(SV40)-3xFLAG-P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9874)
  AUTHORS   Silverstein RA, Kim N, Kroell AS, Walton RT, Delano J, Butcher RM, 
            Pacesa M, Smith BK, Christie KA, Ha LL, Meis RJ, Clark AB, Spinner 
            AD, Lazzarotto CR, Li Y, Matsubara A, Urbina EO, Dahl GA, Correia 
            BE, Marks DS, Tsai SQ, Pinello L, De Ravin SS, Liu Q, Kleinstiver BP
  TITLE     Custom CRISPR-Cas9 PAM variants via scalable engineering and machine
            learning.
  JOURNAL   Nature. 2025 Apr 22. doi: 10.1038/s41586-025-09021-y.
  PUBMED    40262634
REFERENCE   2  (bases 1 to 9874)
  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: "Nature. 
            2025 Apr 22. doi: 10.1038/s41586-025-09021-y."
COMMENT     Sequence Label: pCAG-SpCas9-P2A-EGFP (MSP2582)
FEATURES             Location/Qualifiers
     source          1..9874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          686..1702
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1626..1648
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1710..1729
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1795..1804
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1801..5202,5203..5205,5206..5802,5803,5804..5806,
                     5807..5808,5809..5811,5812..5904)
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1801..5904
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1801..5904
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5911..5931
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5938..6003
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6022..6078
                     /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             join(6079..6081,6082..6084,6085..6798)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6079..6081,6082..6084,6085..6795)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6124..6145)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6385..6404)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6732..6753
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6885..6904)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6950..7005
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7004..7023)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7366..7382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7366..7382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7379..7401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7390..7406
                     /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(7414..7420,7421..7438,7439..7444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7459..7480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7539..7734
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7585..7720
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7740..7874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7777..7796)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7831..7850
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7942..7959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8113..8701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8193..8212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8872..9663,9664..9732))
                     /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     9495..9514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9733..9837)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        join(9868..9874,1..373)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
       61 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      121 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      181 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      241 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      301 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      361 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
      421 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
      481 gggggggggg ggggggcccg ccccgggggg gggggggggg gggggggggg gggggggggg
      541 gggggggggg gggggggggg gggggggggg ggcgaggcgg agaggtgcgg cggcagccaa
      601 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      661 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      721 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      781 gagcgggcgg gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct
      841 tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg
      901 ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc
      961 cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag
     1021 tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg
     1081 gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc
     1141 ggtcgggctg caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc
     1201 gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg
     1261 gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg
     1321 gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct
     1381 tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc
     1441 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc
     1501 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     1561 ccctctccag cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag
     1621 ggcggggttc ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca
     1681 tgccttcttc tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca
     1741 ttttggcaaa gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc
     1801 atggataaaa agtattctat tggtttagac atcggcacta attccgttgg atgggctgtc
     1861 ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt
     1921 cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag
     1981 gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt
     2041 tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt
     2101 ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga
     2161 aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa
     2221 aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat
     2281 atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat
     2341 gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct
     2401 ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga
     2461 cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac
     2521 cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa
     2581 gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca
     2641 caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc
     2701 ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca
     2761 atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt
     2821 cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca
     2881 ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta
     2941 gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga
     3001 aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat
     3061 gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt
     3121 gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct
     3181 cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa
     3241 gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag
     3301 aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg
     3361 tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta
     3421 agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca
     3481 gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc
     3541 tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata
     3601 attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg
     3661 ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct
     3721 cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga
     3781 cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc
     3841 gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac
     3901 tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg
     3961 cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca
     4021 gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta
     4081 atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg
     4141 atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct
     4201 gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg
     4261 gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac
     4321 attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg
     4381 gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag
     4441 aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta
     4501 actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag
     4561 ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat
     4621 acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca
     4681 aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac
     4741 taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa
     4801 tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag
     4861 atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct
     4921 aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga
     4981 cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc
     5041 gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg
     5101 cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc
     5161 gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc
     5221 tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc
     5281 aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac
     5341 ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag
     5401 tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt
     5461 caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc
     5521 cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag
     5581 cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc
     5641 atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa
     5701 cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct
     5761 ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag
     5821 gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata
     5881 gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt ctcgagcgac
     5941 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
     6001 aagggaggcg gtggaagcgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc
     6061 gaagagaatc ctggaccgat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     6121 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     6181 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     6241 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
     6301 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     6361 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     6421 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     6481 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     6541 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     6601 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     6661 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     6721 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     6781 gacgagctgt acaagtgaag cggccgcact cctcaggtgc aggctgccta tcagaaggtg
     6841 gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc
     6901 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     6961 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     7021 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     7081 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     7141 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     7201 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     7261 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     7321 ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     7381 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     7441 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     7501 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     7561 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     7621 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     7681 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     7741 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     7801 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     7861 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     7921 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     7981 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     8041 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     8101 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     8161 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     8221 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     8281 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     8341 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     8401 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     8461 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     8521 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     8581 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     8641 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     8701 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     8761 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     8821 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     8881 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     8941 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     9001 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     9061 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     9121 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     9181 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     9241 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     9301 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     9361 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     9421 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     9481 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     9541 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     9601 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     9661 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     9721 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     9781 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     9841 atttccccga aaagtgccac ctgggtcgac attg
//
