LOCUS       sequence-238647-       14026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCas9-p300 and MS2-p65-HSF1 fusion proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14026)
  AUTHORS   Chong ZS, Ohnishi S, Yusa K, Wright GJ
  TITLE     Pooled extracellular receptor-ligand interaction screening using 
            CRISPR activation.
  JOURNAL   Genome Biol. 2018 Nov 26;19(1):205. doi: 10.1186/s13059-018-1581-3.
  PUBMED    30477585
REFERENCE   2  (bases 1 to 14026)
  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: "Genome 
            Biol."; date: "2018-11-26"; volume: "19(1):205. doi"; pages: " 
            10.1186/s13059-018-1581-3"
COMMENT     Sequence Label: pPB-R1R2_EF1adCas9p300_T2A_MS2p65HSF1-IRESbsdpA
FEATURES             Location/Qualifiers
     source          1..14026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    998..1022
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        1473..2654
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1704..2646
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2603..2623
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             2685..2732
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(2733..2756,2757..2759,2760..5315,5316..5318,
                     5319..6833)
                     /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 N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6888..6908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6930..8780
                     /codon_start=1
                     /gene="EP300"
                     /product="catalytic histone acetyltransferase core of the 
                     human E1A-associated protein p300 (Delvecchio et al., 
                     2013)"
                     /label=p300 core
                     /translation="IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDI
                     VKSPMDLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQ
                     EIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQG
                     ESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAG
                     FVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHA
                     SDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPP
                     PNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDY
                     IFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFE
                     GDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLS
                     RGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDL
                     MDGRDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD"
     regulatory      8544..8553
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8781..8834
                     /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             8844..9230
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             9285..9305
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             9321..9863
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             9888..10259
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     misc_feature    join(10285..10859,10860..10862,10863..10871)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(10452..10469)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     10679..10698
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             10872..11270
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    11274..11498
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(11519..11543)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     repeat_region   11725..11787
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(11838..11858)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(11838..11856)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11877..11893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11877..11893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11890..11912)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11901..11917
                     /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(11925..11931,11932..11949,11950..11955))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11970..11991
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12108..12125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12279..12867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12359..12378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13038..13829,13830..13898))
                     /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     13661..13680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13899..14003)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcaca agtttgtaca aaaaagcagg
     1021 ctggcgccgg aaccaattca gtcgactgga tccacgcgtg aggggcgcgg aagccggggc
     1081 ggaggaggag ccaaccccgg gctgggctga gacccgcaga ggaagacgct ctagggattt
     1141 gtcccggact agcgagatgg caaggctgag gacgggaggc tgattgagag gcgaaggtac
     1201 accctaatct caatacaacc tttggagcta agccagcaat ggtagaggga agattctgca
     1261 cgtcccttcc aggcggcctc cccgtcacca ccccccccaa cccgccccga ccggagctga
     1321 gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa
     1381 ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc
     1441 gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc
     1501 gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct
     1561 agagaaagtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc
     1621 ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca
     1681 acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
     1741 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg
     1801 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
     1861 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
     1921 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
     1981 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
     2041 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
     2101 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
     2161 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
     2221 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     2281 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     2341 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     2401 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     2461 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     2521 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     2581 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     2641 tttcaggtgt cgtggaagct tggcgcgccc gtacggccac catgaaaagg ccggcggcca
     2701 cgaaaaaggc cggccaggca aaaaagaaaa aggacaagaa gtacagcatc ggcctggcca
     2761 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     2821 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     2881 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     2941 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     3001 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     3061 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     3121 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     3181 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     3241 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     3301 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     3361 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     3421 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     3481 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     3541 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     3601 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     3661 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     3721 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     3781 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     3841 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     3901 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     3961 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     4021 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     4081 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     4141 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     4201 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     4261 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     4321 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     4381 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     4441 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     4501 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     4561 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     4621 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     4681 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     4741 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     4801 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     4861 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     4921 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     4981 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     5041 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     5101 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     5161 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     5221 accggctgtc cgactacgat gtggaccaca tcgtgcctca gagctttctg aaggacgact
     5281 ccatcgacaa caaggtgctg accagaagcg acaaggcccg gggcaagagc gacaacgtgc
     5341 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     5401 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     5461 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     5521 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     5581 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     5641 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     5701 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     5761 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     5821 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     5881 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     5941 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     6001 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     6061 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     6121 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     6181 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     6241 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     6301 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     6361 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     6421 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     6481 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     6541 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     6601 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     6661 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     6721 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     6781 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacagcgctg
     6841 gaggaggtgg aagcggagga ggaggaagcg gaggaggagg tagcggacct aagaaaaaga
     6901 ggaaggtggc gggaggtgga agcggaggaa ttttcaaacc agaagaacta cgacaggcac
     6961 tgatgccaac tttggaggca ctttaccgtc aggatccaga atcccttccc tttcgtcaac
     7021 ctgtggaccc tcagctttta ggaatccctg attactttga tattgtgaag agccccatgg
     7081 atctttctac cattaagagg aagttagaca ctggacagta tcaggagccc tggcagtatg
     7141 tcgatgatat ttggcttatg ttcaataatg cctggttata taaccggaaa acatcacggg
     7201 tatacaaata ctgctccaag ctctctgagg tctttgaaca agaaattgac ccagtgatgc
     7261 aaagccttgg atactgttgt ggcagaaagt tggagttctc tccacagaca ctgtgttgct
     7321 acggcaaaca gttgtgcaca atacctcgtg atgccactta ttacagttac cagaacaggt
     7381 atcatttctg tgagaagtgt ttcaatgaga tccaagggga gagcgtttct ttgggggatg
     7441 acccttccca gcctcaaact acaataaata aagaacaatt ttccaagaga aaaaatgaca
     7501 cactggatcc tgaactgttt gttgaatgta cagagtgcgg aagaaagatg catcagatct
     7561 gtgtccttca ccatgagatc atctggcctg ctggattcgt ctgtgatggc tgtttaaaga
     7621 aaagtgcacg aactaggaaa gaaaataagt tttctgctaa aaggttgcca tctaccagac
     7681 ttggcacctt tcttgagaat cgtgtgaatg actttctgag gcgacagaat caccctgagt
     7741 caggagaggt cactgttaga gtagttcatg cttctgacaa aaccgtggaa gtaaaaccag
     7801 gcatgaaagc aaggtttgtg gacagtggag agatggcaga atcctttcca taccgaacca
     7861 aagccctctt tgcctttgaa gaaattgatg gtgttgacct gtgcttcttt ggcatgcatg
     7921 ttcaagagta tggctctgac tgccctccac ccaaccagag gagagtatac atatcttacc
     7981 tcgatagtgt tcatttcttc cgtcctaaat gcttgaggac tgcagtctat catgaaatcc
     8041 taattggata tttagaatat gtcaagaaat taggttacac aacagggcat atttgggcat
     8101 gtccaccaag tgagggagat gattatatct tccattgcca tcctcctgac cagaagatac
     8161 ccaagcccaa gcgactgcag gaatggtaca aaaaaatgct tgacaaggct gtatcagagc
     8221 gtattgtcca tgactacaag gatattttta aacaagctac tgaagataga ttaacaagtg
     8281 caaaggaatt gccttatttc gagggtgatt tctggcccaa tgttctggaa gaaagcatta
     8341 aggaactgga acaggaggaa gaagagagaa aacgagagga aaacaccagc aatgaaagca
     8401 cagatgtgac caagggagac agcaaaaatg ctaaaaagaa gaataataag aaaaccagca
     8461 aaaataagag cagcctgagt aggggcaaca agaagaaacc cgggatgccc aatgtatcta
     8521 acgacctctc acagaaacta tatgccacca tggagaagca taaagaggtc ttctttgtga
     8581 tccgcctcat tgctggccct gctgccaact ccctgcctcc cattgttgat cctgatcctc
     8641 tcatcccctg cgatctgatg gatggtcggg atgcgtttct cacgctggca agggacaagc
     8701 acctggagtt ctcttcactc cgaagagccc agtggtccac catgtgcatg ctggtggagc
     8761 tgcacacgca gagccaggac gagggcagag gaagtctcct aacatgcggt gacgtggagg
     8821 agaatcctgg cccaggaggt atggcttcaa actttactca gttcgtgctc gtggacaatg
     8881 gtgggacagg ggatgtgaca gtggctcctt ctaatttcgc taatggggtg gcagagtgga
     8941 tcagctccaa ctcacggagc caggcctaca aggtgacatg cagcgtcagg cagtctagtg
     9001 cccagaagag aaagtatacc atcaaggtgg aggtccccaa agtggctacc cagacagtgg
     9061 gcggagtcga actgcctgtc gccgcttgga ggtcctacct gaacatggag ctcactatcc
     9121 caattttcgc taccaattct gactgtgaac tcatcgtgaa ggcaatgcag gggctcctca
     9181 aagacggtaa tcctatccct tccgccatcg ccgctaactc aggtatctac agcgctggag
     9241 gaggtggaag cggaggagga ggaagcggag gaggaggtag cggacctaag aaaaagagga
     9301 aggtggcggc cgctggatcc ccttcagggc agatcagcaa ccaggccctg gctctggccc
     9361 ctagctccgc tccagtgctg gcccagacta tggtgccctc tagtgctatg gtgcctctgg
     9421 cccagccacc tgctccagcc cctgtgctga ccccaggacc accccagtca ctgagcgctc
     9481 cagtgcccaa gtctacacag gccggcgagg ggactctgag tgaagctctg ctgcacctgc
     9541 agttcgacgc tgatgaggac ctgggagctc tgctggggaa cagcaccgat cccggagtgt
     9601 tcacagatct ggcctccgtg gacaactctg agtttcagca gctgctgaat cagggcgtgt
     9661 ccatgtctca tagtacagcc gaaccaatgc tgatggagta ccccgaagcc attacccggc
     9721 tggtgaccgg cagccagcgg ccccccgacc ccgctccaac tcccctggga accagcggcc
     9781 tgcctaatgg gctgtccgga gatgaagact tctcaagcat cgctgatatg gactttagtg
     9841 ccctgctgtc acagatttcc tctagtgggc agggaggagg tggaagcggc ttcagcgtgg
     9901 acaccagtgc cctgctggac ctgttcagcc cctcggtgac cgtgcccgac atgagcctgc
     9961 ctgaccttga cagcagcctg gccagtatcc aagagctcct gtctccccag gagcccccca
    10021 ggcctcccga ggcagagaac agcagcccgg attcagggaa gcagctggtg cactacacag
    10081 cgcagccgct gttcctgctg gaccccggct ccgtggacac cgggagcaac gacctgccgg
    10141 tgctgtttga gctgggagag ggctcctact tctccgaagg ggacggcttc gccgaggacc
    10201 ccaccatctc cctgctgaca ggctcggagc ctcccaaagc caaggacccc actgtctcct
    10261 gtacatgagg aattccgccc cccccccctc tccctccccc ccccctaacg ttactggccg
    10321 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
    10381 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
    10441 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
    10501 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
    10561 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
    10621 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
    10681 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
    10741 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
    10801 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataata
    10861 tggccacaac catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa
    10921 cggctacaat caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct
    10981 ctagcgacgg ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg
    11041 cagaactcgt ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg
    11101 tcgcgatcgg aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc
    11161 ttctcgatct gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg
    11221 cagttgggat tcgtgaattg ctgccctctg gttatgtgtg ggagggctga cgactgtgcc
    11281 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
    11341 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
    11401 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaagt
    11461 caatagcagg catgctgggg atgcggtggg ctctatggac tagtctcgag atatctagac
    11521 ccagctttct tgtacaaagt ggtactagtt aaaagttttg ttactttata gaagaaattt
    11581 tgagtttttg ttttttttta ataaataaat aaacataaat aaattgtttg ttgaatttat
    11641 tattagtatg taagtgtaaa tataataaaa cttaatatct attcaaatta ataaataaac
    11701 ctcgatatac agaccgataa aacacatgcg tcaattttac gcatgattat ctttaacgta
    11761 cgtcacaata tgattatctt tctagggtta atctagtata cgcgtatgcg gccgcttaat
    11821 taatccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
    11881 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
    11941 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
    12001 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
    12061 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
    12121 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
    12181 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
    12241 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
    12301 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
    12361 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
    12421 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
    12481 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
    12541 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
    12601 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
    12661 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
    12721 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
    12781 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
    12841 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
    12901 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
    12961 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
    13021 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
    13081 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
    13141 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
    13201 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
    13261 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
    13321 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
    13381 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
    13441 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
    13501 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
    13561 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
    13621 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
    13681 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
    13741 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
    13801 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
    13861 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
    13921 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
    13981 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
