LOCUS       sequence-354375-        9936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCas9 fused to P65.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9936)
  AUTHORS   Tak YE, Horng JE, Perry NT, Schultz HT, Iyer S, Yao Q, Zou LS, Aryee
            MJ, Pinello L, Joung JK
  TITLE     Augmenting and directing long-range CRISPR-mediated activation in 
            human cells.
  JOURNAL   Nat Methods. 2021 Sep;18(9):1075-1081. doi: 
            10.1038/s41592-021-01224-1. Epub 2021 Aug 5.
  PUBMED    34354266
REFERENCE   2  (bases 1 to 9936)
  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"; doi: 
            "10.1038/s41592-021-01224-1"; journalName: "Nat Methods"; date: 
            "2021-09"; volume: "18"; issue: "9"; pages: "1075-1081"
COMMENT     Sequence Label: BPK1160
FEATURES             Location/Qualifiers
     source          1..9936
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1772..1781
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1784..1804
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1841..5239,5240..5242,5243..5839,5840,5841..5843,
                     5844..5845,5846..5848,5849..5941)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1841..1864,1865..1867,1868..4354,4355..4357,
                     4358..5941)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5948..5968
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5975..6040
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6497..6868
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     primer_bind     complement(6958..6977)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    7023..7078
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(7077..7096)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(7439..7455)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7439..7455)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7452..7474)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7463..7479
                     /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(7487..7493,7494..7511,7512..7517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7532..7553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7612..7807
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      7658..7793
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    7813..7947
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7850..7869)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7904..7923
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8015..8032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8186..8774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8266..8285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8945..9736,9737..9805))
                     /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     9568..9587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9806..9910)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggnsgcg cgccrggsgg ggsggggsgg ggsgrggggs
      541 ggggsggggs grggsggrga ggkgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttctgcagtc
     1741 gacggtaccg cgggcccggg atccaccggt cgccaccatg gcgccgaaaa aaaaacgcaa
     1801 agtgaacggc ggagggtccg gaggaggcgg tagcggaggc gataaaaagt attctattgg
     1861 tttagccatc ggcactaatt ccgttggatg ggctgtcata accgatgaat acaaagtacc
     1921 ttcaaagaaa tttaaggtgt tggggaacac agaccgtcat tcgattaaaa agaatcttat
     1981 cggtgccctc ctattcgata gtggcgaaac ggcagaggcg actcgcctga aacgaaccgc
     2041 tcggagaagg tatacacgtc gcaagaaccg aatatgttac ttacaagaaa tttttagcaa
     2101 tgagatggcc aaagttgacg attctttctt tcaccgtttg gaagagtcct tccttgtcga
     2161 agaggacaag aaacatgaac ggcaccccat ctttggaaac atagtagatg aggtggcata
     2221 tcatgaaaag tacccaacga tttatcacct cagaaaaaag ctagttgact caactgataa
     2281 agcggacctg aggttaatct acttggctct tgcccatatg ataaagttcc gtgggcactt
     2341 tctcattgag ggtgatctaa atccggacaa ctcggatgtc gacaaactgt tcatccagtt
     2401 agtacaaacc tataatcagt tgtttgaaga gaaccctata aatgcaagtg gcgtggatgc
     2461 gaaggctatt cttagcgccc gcctctctaa atcccgacgg ctagaaaacc tgatcgcaca
     2521 attacccgga gagaagaaaa atgggttgtt cggtaacctt atagcgctct cactaggcct
     2581 gacaccaaat tttaagtcga acttcgactt agctgaagat gccaaattgc agcttagtaa
     2641 ggacacgtac gatgacgatc tcgacaatct actggcacaa attggagatc agtatgcgga
     2701 cttatttttg gctgccaaaa accttagcga tgcaatcctc ctatctgaca tactgagagt
     2761 taatactgag attaccaagg cgccgttatc cgcttcaatg atcaaaaggt acgatgaaca
     2821 tcaccaagac ttgacacttc tcaaggccct agtccgtcag caactgcctg agaaatataa
     2881 ggaaatattc tttgatcagt cgaaaaacgg gtacgcaggt tatattgacg gcggagcgag
     2941 tcaagaggaa ttctacaagt ttatcaaacc catattagag aagatggatg ggacggaaga
     3001 gttgcttgta aaactcaatc gcgaagatct actgcgaaag cagcggactt tcgacaacgg
     3061 tagcattcca catcaaatcc acttaggcga attgcatgct atacttagaa ggcaggagga
     3121 tttttatccg ttcctcaaag acaatcgtga aaagattgag aaaatcctaa cctttcgcat
     3181 accttactat gtgggacccc tggcccgagg gaactctcgg ttcgcatgga tgacaagaaa
     3241 gtccgaagaa acgattactc catggaattt tgaggaagtt gtcgataaag gtgcgtcagc
     3301 tcaatcgttc atcgagagga tgaccaactt tgacaagaat ttaccgaacg aaaaagtatt
     3361 gcctaagcac agtttacttt acgagtattt cacagtgtac aatgaactca cgaaagttaa
     3421 gtatgtcact gagggcatgc gtaaacccgc ctttctaagc ggagaacaga agaaagcaat
     3481 agtagatctg ttattcaaga ccaaccgcaa agtgacagtt aagcaattga aagaggacta
     3541 ctttaagaaa attgaatgct tcgattctgt cgagatctcc ggggtagaag atcgatttaa
     3601 tgcgtcactt ggtacgtatc atgacctcct aaagataatt aaagataagg acttcctgga
     3661 taacgaagag aatgaagata tcttagaaga tatagtgttg actcttaccc tctttgaaga
     3721 tcgggaaatg attgaggaaa gactaaaaac atacgctcac ctgttcgacg ataaggttat
     3781 gaaacagtta aagaggcgtc gctatacggg ctggggacga ttgtcgcgga aacttatcaa
     3841 cgggataaga gacaagcaaa gtggtaaaac tattctcgat tttctaaaga gcgacggctt
     3901 cgccaatagg aactttatgc agctgatcca tgatgactct ttaaccttca aagaggatat
     3961 acaaaaggca caggtttccg gacaagggga ctcattgcac gaacatattg cgaatcttgc
     4021 tggttcgcca gccatcaaaa agggcatact ccagacagtc aaagtagtgg atgagctagt
     4081 taaggtcatg ggacgtcaca aaccggaaaa cattgtaatc gagatggcac gcgaaaatca
     4141 aacgactcag aaggggcaaa aaaacagtcg agagcggatg aagagaatag aagagggtat
     4201 taaagaactg ggcagccaga tcttaaagga gcatcctgtg gaaaataccc aattgcagaa
     4261 cgagaaactt tacctctatt acctacaaaa tggaagggac atgtatgttg atcaggaact
     4321 ggacataaac cgtttatctg attacgacgt cgatgccatt gtaccccaat cctttttgaa
     4381 ggacgattca atcgacaata aagtgcttac acgctcggat aagaaccgag ggaaaagtga
     4441 caatgttcca agcgaggaag tcgtaaagaa aatgaagaac tattggcggc agctcctaaa
     4501 tgcgaaactg ataacgcaaa gaaagttcga taacttaact aaagctgaga ggggtggctt
     4561 gtctgaactt gacaaggccg gatttattaa acgtcagctc gtggaaaccc gccaaatcac
     4621 aaagcatgtt gcacagatac tagattcccg aatgaatacg aaatacgacg agaacgataa
     4681 gctgattcgg gaagtcaaag taatcacttt aaagtcaaaa ttggtgtcgg acttcagaaa
     4741 ggattttcaa ttctataaag ttagggagat aaataactac caccatgcgc acgacgctta
     4801 tcttaatgcc gtcgtaggga ccgcactcat taagaaatac ccgaagctag aaagtgagtt
     4861 tgtgtatggt gattacaaag tttatgacgt ccgtaagatg atcgcgaaaa gcgaacagga
     4921 gataggcaag gctacagcca aatacttctt ttattctaac attatgaatt tctttaagac
     4981 ggaaatcact ctggcaaacg gagagatacg caaacgacct ttaattgaaa ccaatgggga
     5041 gacaggtgaa atcgtatggg ataagggccg ggacttcgcg acggtgagaa aagttttgtc
     5101 catgccccaa gtcaacatag taaagaaaac tgaggtgcag accggagggt tttcaaagga
     5161 atcgattctt ccaaaaagga atagtgataa gctcatcgct cgtaaaaagg actgggaccc
     5221 gaaaaagtac ggtggcttcg atagccctac agttgcctat tctgtcctag tagtggcaaa
     5281 agttgagaag ggaaaatcca agaaactgaa gtcagtcaaa gaattattgg ggataacgat
     5341 tatggagcgc tcgtcttttg aaaagaaccc catcgacttc cttgaggcga aaggttacaa
     5401 ggaagtaaaa aaggatctca taattaaact accaaagtat agtctgtttg agttagaaaa
     5461 tggccgaaaa cggatgttgg ctagcgccgg agagcttcaa aaggggaacg aactcgcact
     5521 accgtctaaa tacgtgaatt tcctgtattt agcgtcccat tacgagaagt tgaaaggttc
     5581 acctgaagat aacgaacaga agcaactttt tgttgagcag cacaaacatt atctcgacga
     5641 aatcatagag caaatttcgg aattcagtaa gagagtcatc ctagctgatg ccaatctgga
     5701 caaagtatta agcgcataca acaagcacag ggataaaccc atacgtgagc aggcggaaaa
     5761 tattatccat ttgtttactc ttaccaacct cggcgctcca gccgcattca agtattttga
     5821 cacaacgata gatcgcaaac gatacacttc taccaaggag gtgctagacg cgacactgat
     5881 tcaccaatcc atcacgggat tatatgaaac tcggatagat ttgtcacagc ttgggggtga
     5941 cggatccccc aagaagaaga ggaaagtctc gagcgactac aaagaccatg acggtgatta
     6001 taaagatcat gacatcgatt acaaggatga cgatgacaag gctgcaggag gcggtggaag
     6061 cgggatggag ttccagtacc tgccagatac agacgatcgt caccggattg aggagaaacg
     6121 taaaaggaca tatgagacct tcaagagcat catgaagaag agtcctttca gcggacccac
     6181 cgacccccgg cctccacctc gacgcattgc tgtgccttcc cgcagctcag cttctgtccc
     6241 caagccagca ccccagccct atccctttac gtcatccctg agcaccatca actatgatga
     6301 gtttcccacc atggtgtttc cttctgggca gatcagccag gcctcggcct tggccccggc
     6361 ccctccccaa gtcctgcccc aggctccagc ccctgcccct gctccagcca tggtatcagc
     6421 tctggcccag gccccagccc ctgtcccagt cctagcccca ggccctcctc aggctgtggc
     6481 cccacctgcc cccaagccca cccaggctgg ggaaggaacg ctgtcagagg ccctgctgca
     6541 gctgcagttt gatgatgaag acctgggggc cttgcttggc aacagcacag acccagctgt
     6601 gttcacagac ctggcatccg tcgataactc cgagtttcag cagctgctga accagggcat
     6661 acctgtggcc ccccacacaa ctgagcccat gctgatggag taccctgagg ctataactcg
     6721 cctagtgaca ggggcccaga ggccccccga cccagctcct gctccactgg gggccccggg
     6781 gctccccaat ggcctccttt caggagatga agacttctcc tccattgcgg acatggactt
     6841 ctcagccctg ctgagtcaga tcagctctta aagcggccgc actcctcagg tgcaggctgc
     6901 ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc actgagatct
     6961 ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc tgacttctgg
     7021 ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact
     7081 cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt tggtttagag
     7141 tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat aaagaggtca
     7201 tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag ccttgacttg
     7261 aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat ccctaaaatt
     7321 ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc cagtcatagc
     7381 tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggcg taatcatggt
     7441 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     7501 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     7561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa
     7621 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     7681 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     7741 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     7801 ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     7861 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     7921 atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga
     7981 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     8041 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     8101 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     8161 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     8221 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     8281 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     8341 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     8401 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     8461 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     8521 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     8581 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     8641 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     8701 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     8761 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     8821 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     8881 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     8941 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     9001 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     9061 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     9121 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     9181 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     9241 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     9301 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     9361 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     9421 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     9481 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     9541 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     9601 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     9661 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     9721 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     9781 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     9841 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     9901 gggttccgcg cacatttccc cgaaaagtgc cacctg
//
