LOCUS       sequence-154056-       15540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3XFLAG-dCas9-VPR (human codon) under 10XUAS control, for 
            in vivo CRISPRa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15540)
  AUTHORS   Lin S, Ewen-Campen B, Ni X, Housden BE, Perrimon N
  TITLE     In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.
  JOURNAL   Genetics. 2015 Oct;201(2):433-42. doi: 10.1534/genetics.115.181065. 
            Epub 2015 Aug 5.
  PUBMED    26245833
REFERENCE   2  (bases 1 to 15540)
  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: 
            "10.1534/genetics.115.181065"; journalName: "Genetics"; date: 
            "2015-10"; volume: "201"; issue: "2"; pages: "433-42"
COMMENT     Sequence Label: pWalium20-10XUAS-3XFLAG-dCas9-VPR
FEATURES             Location/Qualifiers
     source          1..15540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(33..461)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     355..374
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     588..610
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     602..619
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     603..619
                     /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"
     CDS             complement(join(1444..2058,2129..2260,2481..2796,2858..3512,
                     3587..3860,4255..4326))
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLLPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     primer_bind     4786..4804
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     protein_bind    4923..4992
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     protein_bind    5230..5660
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     protein_bind    5678..5711
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5724..5818
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     protein_bind    5834..5867
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5880..5974
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     promoter        5998..6236
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     6183..6204
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     regulatory      6321..6330
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6330..6395
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6402..6422
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6447..6470,6471..6473,6474..8957,8958..8963,8964..9029,
                     9030..9032,9033..10547)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             10560..10580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             10605..10754
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             10779..10799
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11229..11585
                     /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
                     EDFSSIADMDFSALL"
     CDS             11604..12173
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     intron          12276..12341
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             12471..12491
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    12763..12884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(12800..12819)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     12854..12873
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    12906..13336
                     /label=gypsy insulator
                     /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2"
                     /note="chromatin insulator from Drosophila"
     promoter        complement(13355..13373)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(13394..13410)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13394..13410)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13407..13429)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13418..13434
                     /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(13442..13448,13449..13466,13467..13472))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13487..13508
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13625..13642)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13796..14384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13876..13895)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14555..15346,15347..15415))
                     /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     15178..15197
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15416..15520)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
      361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
      421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
      481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtacaag
      661 cttcacgacc tgaggcgcgc ctccagtgaa atccaagcat tttctaaatt aaatgtattc
      721 ttattattat agttgttatt tttgatatat ataaacaaca ctattatgcc caccattttt
      781 ttgagatgca tctacacaag gaacaaacac tggatgtcac tttcagttca aattgtaacg
      841 ctaatcactc cgaacaggtc acaaaaaatt accttaaaaa gtcataatat taaattagaa
      901 taaatatagc tgtgagggaa atatatacaa atatattgga gcaaataaat tgtacataca
      961 aatatttatt actaatttct attgagacga aatgaaccac tcggaaccat ttgagcgaac
     1021 cgaatcgcgc ggaactaacg acagtcgctc caaggtcgtc gaacaaaagg tgaatgtgtt
     1081 gcggagagcg ggtgggagac agcgaaagag caactacgaa acgtggtgtg gtggaggtga
     1141 attatgaaga gggcgcgcga tttgaaaagt atgtatataa aaaatatatc ccggtgtttt
     1201 atgtagcgat aaacgagttt ttgatgtaag gtatgcaggt gtgtaagtct tttggttaga
     1261 agacaaatcc aaagtctact tgtggggatg ttcgaagggg aaatacttgt attctatagg
     1321 tcatatcttg tttttattgg cacaaatata attacattag ctttttgagg gggcaataaa
     1381 cagtaaacac gatggtaata atggtaaaaa aaaaaacaag cagttatttc ggatatatgt
     1441 cggctactcc ttgcgtcggg cccgaagtct tagagccaga tatgcgagca cccggaagct
     1501 cacgatgaga atggccagac ccacgtagtc cagcggcaga tcggcggcgg agaagttaag
     1561 cgtctccagg atgaccttgc ccgaactggg gcacgtggtg ttcgacgatg tgcagctaat
     1621 ttcgcccggc tccacgtccg cccattggtt aatcagcaga ccctcgttgg cgtaacggaa
     1681 ccatgagagg tacgacaacc atttgaggta tactggcacc gagcccgagt tcaagaagaa
     1741 gccgccaaag agcaggaatg gtatgataac cggcggaccc acagacagcg ccatcgaggt
     1801 cgaggagctg gcgcaggata ttagatatcc gaaggacgtt gacacattgg ccaccagagt
     1861 gaccagcgcc aggcagttga agaagtgcag cactccggcc cgcagtccga tcatcggata
     1921 ggcaatcgcc gtgaagacca gtggcactgt gagaaaaagc ggtaattcgg caatcgtttt
     1981 gcccagaaag tatgtgtcac agcgataaag tcgacttcgg gcctccctca taaaaactgg
     2041 cagctctgag gtgaacacct aaatcgaatc gattcattag aaagttagta aattattaat
     2101 atgcaaatgt attctaaaca agacttacat ttatcgtggc aaagacgttt tgaaaggtca
     2161 tgttggtcag gaagaggaag atggctccgt tgatattcat cacgcccact tgcgtgagtt
     2221 gttggcccaa aaagatgagg ccaatcaaga tggcaaccat ctgcaaatta aaatgttact
     2281 cgcatctcat taatattcat atcttcaaca tgttcgcgag ttaaatgaaa tttatttatt
     2341 ttctgcaaaa ctataaacta tacatctcat tgaaaaaaac taagaagggt gtggaatcag
     2401 gcaattctaa ctaaaatcta gcgaatttgt ttccaagaat tgtaagcgtt atatcatttg
     2461 tttccactgg aaccactcac cgttgtctga ataagtcgca cttttacgag gagtggttcc
     2521 ttgagcaccg acagccagga tcgccacagg accgcccgga actgcatgaa ccaggtggcc
     2581 ttgtaggtgt acccattctc cggctgctcc agtggcttct ccaaattttt ggtggccaac
     2641 aactgctcca tatcccgggc tactttgcta atagcaaaat tgtcgcatat cttggcgatc
     2701 cgatcacggg actcgatctc ccgtccgggc acaacggcca acacctgtac gtaaaagtcc
     2761 gccggattgt agttggtagg acactgggca cccacgctgg ataggagttg agatgtaatg
     2821 taatgctaga tacccttaat aaacacatcg aactcactag gaaaagaagt cgacggcttc
     2881 gctgggagtg cccaagaaag ctaccctgcc ctcggccatc agaaggatct tgtcaaagag
     2941 ctcaaacagc tcggaagacg gctgatgaat ggtcaggatg acggtcttgc ccttctgcga
     3001 cagcttcttc agcacctgga cgacgctgtg ggcggtaaat gagtccagtc cggaggtggg
     3061 ctcatcgcag atcagaagcg gcggatcggt tagtgcctcg gaggcgaatg ccagacgctt
     3121 cctttctccg ccggacagac ctttcaccct gccgggcaca ccgatgatcg tgtgctgaca
     3181 tttgctgagc gaaagctcct ggatcacctg atccacgcgg gccactcgct gccgataggt
     3241 cagatgtcgt ggcatccgca ccatggcctg gaaaatcagg tgttccctgg ccgttaggga
     3301 gccgataaag aggtcatcct gctggacata ggcgcacctg gcctgcatct ccttggcgtc
     3361 cacaggttgg ccattgagca gtcgcatccc ggatggcgat acttggatgc cctgcggcga
     3421 tcgaaaggca agggcattca gcagggtcgt ctttccggca ccggaactgc ccatcacggc
     3481 caaaagttcg cccggatagg ccacgccgca aactgagttt caaattggta attggaccct
     3541 ttattaagat ttcacacaga tcagccgact gcgaatagaa actcaccgtt cttgagcaaa
     3601 tgtttcctgg gcgccggtat gtgtcgctcg ttgcagaata gtccgcgtgt ccggttgacc
     3661 agctgccgcc atccggagcc cggctgattg accgccccaa agatgtccat attgtgccag
     3721 gcataggtga ggttctcggc tagttggccg ctccctgaac cggagtcctc cggcggactg
     3781 ggtggcagga gcgtgccgta gtttttggcc tgcccgaagc cctggttaat gcagctctgc
     3841 gaagccgctc cgctgtcacc ctgcaatgat aggggatctc aaatatcaac tacaagcgtt
     3901 atgctcatct aaccccgaac aaaacgaagt atcctacgaa gtaggtttat acttttattt
     3961 attttttgtg catctaggat cagcttaaaa tatctggttg ttatattttt tgtaaaaaag
     4021 aatgtagtcg aaaatgaatg cctttagatg tcttgatcat gatatgatct taaaaattgt
     4081 cttatatagc gagcacagct accagaataa tctgtttcgt gtcactattt gtttgtgcga
     4141 ttgcggtttg ggatttttgt gggtcgcagt tctcacgccg cagacaattt gatgttgcaa
     4201 tcgcagttcc tatagatcaa gtgaacttaa gatgtatgca catgtactac tcacattgtt
     4261 cagatgctcg gcagatgggt gtttgctgcc tccgcgaatt aatagctcct gatcctcttg
     4321 gcccattgcc gggatttttc acactttccc ctgcttaccc acccaaaacc aatcaccacc
     4381 ccaatcactc aaaaaacaaa caaaaataag aagcgagagg agttttggca cagcactttg
     4441 tgtttaattg atggcgtaaa ccgcttggag cttcgtcacg aaaccgctga caaagtgcaa
     4501 ctgaaggcgg acattgacgc taggtaacgc tacaaacggt ggcgaaagag atagcggacg
     4561 cagcggcgaa agagacggcg atatttctgt ggacagagaa ggaggcaaac agcgctgact
     4621 ttgagtggaa tgtcattttg agtgagaggt aatcgaaaga acctggtact tcaaataccc
     4681 ttggatcgaa gtaaatttaa aactgatcag ataagttcaa tgatatccag tgcagtaaaa
     4741 aataaaaaaa aaaaatgttt ttttttatct actttccgca aaaatgggtt ttattaactt
     4801 acatacatgg cgcgccagat cgcaagaagc ttgatatcat cgatctcgag gctgcatcca
     4861 acgcgttggg agctctccgg atcaattcgg cttcaggtac cgtcgacgat gtaggtcacg
     4921 gtctcgaagc cgcggtgcgg gtgccagggc gtgcccttgg gctccccggg cgcgtactcc
     4981 acctcaccca tctggtccat catgatgaac gggtcgaggt ggcggtagtt gatcccggcg
     5041 aacgcgcggc gcaccgggaa gccctcgccc tcgaaaccgc tgggcgcggt ggtcacggtg
     5101 agcacgggac gtgcgacggc gtcggcgggt gcggatacgc ggggcagcgt cagcgggttc
     5161 tcgacggtca cggcgggcat gtcgacaagc cgaattgatc cactagaagg cctaattcgg
     5221 tacactagtt ggccacgtaa taagtgtgcg ttgaatttat tcgcaaaaac attgcatatt
     5281 ttcggcaaag taaaattttg ttgcatacct tatcaaaaaa taagtgctgc atacttttta
     5341 gagaaaccaa ataatttttt attgcatacc cgtttttaat aaaatacatt gcataccctc
     5401 ttttaataaa aaatattgca tactttgacg aaacaaattt tcgttgcata cccaataaaa
     5461 gattattata ttgcataccc gtttttaata aaatacattg cataccctct tttaataaaa
     5521 aatattgcat acgttgacga aacaaatttt cgttgcatac ccaataaaag attattatat
     5581 tgcatacctt ttcttgccat accatttagc cgatcaattg tgctcggcaa cagtatattt
     5641 gtggtgtgcc aaccaacaac actagtagta ccagcttata acttcgtata atgtatgcta
     5701 tacgaagtta tctgcaggca ggtcggagta ctgtcctccg agcggagtac tgtcctccga
     5761 gcggagtact gtcctccgag cggagtactg tcctccgagc ggagtactgt cctccgagcg
     5821 gagactccca tggataactt cgtataatgt atgctatacg aagttatgga tccgcaggtc
     5881 ggagtactgt cctccgagcg gagtactgtc ctccgagcgg agtactgtcc tccgagcgga
     5941 gtactgtcct ccgagcggag tactgtcctc cgagcggaga ctcgtcgaca gcgagcgccg
     6001 gagtataaat agaggcgctt cgtctacgga gcgacaattc aattcaaaca agcaaagtga
     6061 acacgtcgct aagcgaaagc taagcaaata aacaagcgca gctgaacaag ctaaacaatc
     6121 tgcagtaaag tgcaagttaa agtgaatcaa ttaaaagtaa ccagcaacca agtaaatcaa
     6181 ctgcaactac tgaaatctgc caagaagtaa ttattgaata caagaagaga actctgaata
     6241 gggaattggg aattgagatc tgttctagaa aacatcccat aaaacatccc atattcagcc
     6301 gctagcatgg atgttttccc gccaccatgg attacaagga tcacgatggc gactacaagg
     6361 accatgacat cgactataag gacgacgacg ataagatggc ccccaagaag aagcgcaaag
     6421 tgggcatcca tggcgtgcca gccgccgaca agaagtactc cattgggctc gctatcggca
     6481 caaacagcgt cggctgggcc gtcattacgg acgagtacaa ggtgccgagc aaaaaattca
     6541 aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc gccctcctgt
     6601 tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg cgcagatata
     6661 cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag atggctaagg
     6721 tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag gataaaaagc
     6781 acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc
     6841 caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct gacttgcggt
     6901 tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc atcgaggggg
     6961 acctgaaccc agacaacagc gatgtcgata aactctttat ccaactggtt cagacttaca
     7021 atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa gcaatcctga
     7081 gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc cctggggaga
     7141 agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc cccaacttta
     7201 aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac acctacgatg
     7261 atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt tttttggcgg
     7321 caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac acggagatca
     7381 ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac caagacttga
     7441 ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa attttcttcg
     7501 atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag gaggaatttt
     7561 acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc
     7621 ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc atcccccacc
     7681 agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc tacccctttt
     7741 tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc tactatgtag
     7801 gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca gaagagacca
     7861 tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag tccttcatcg
     7921 aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc
     7981 tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac gtcacagaag
     8041 ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg gacctcctct
     8101 tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc aaaaagattg
     8161 aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca tccctgggaa
     8221 cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat gaggagaacg
     8281 aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg gagatgattg
     8341 aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa cagctcaaga
     8401 ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg atccgagaca
     8461 agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc aaccggaact
     8521 tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag aaagcacaag
     8581 tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt agcccagcta
     8641 tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa gtaatgggaa
     8701 ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact acccagaagg
     8761 gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa gaactggggt
     8821 cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag aagctctacc
     8881 tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac atcaatcggc
     8941 tctccgacta cgacgtggct gctatcgtgc cccagtcttt tctcaaagat gattctattg
     9001 ataataaagt gttgacaaga tccgataaag ctagagggaa gagtgataac gtcccctcag
     9061 aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc aaactgatca
     9121 cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct gagttggata
     9181 aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag cacgtggccc
     9241 aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg attcgagagg
     9301 tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac tttcagtttt
     9361 ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg aatgcagtgg
     9421 taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt tacggagact
     9481 ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata ggcaaggcca
     9541 ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag attacactgg
     9601 ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg
     9661 tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga
     9721 acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt atcctcccga
     9781 aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag aaatacggcg
     9841 gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg gagaaaggga
     9901 agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa
     9961 gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag gtcaaaaaag
    10021 acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc cggaaacgaa
    10081 tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc tctaaatacg
    10141 ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc gaagataatg
    10201 agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc atcgagcaaa
    10261 taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag gtgctttctg
    10321 cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt atccacttgt
    10381 ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc accatagaca
    10441 gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat cagtcaatta
    10501 cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacagc agggctgacc
    10561 ccaagaagaa gaggaaggtg gaggccagcg gttccggacg ggctgacgca ttggacgatt
    10621 ttgatctgga tatgctggga agtgacgccc tcgatgattt tgaccttgac atgcttggtt
    10681 cggatgccct tgatgacttt gacctcgaca tgctcggcag tgacgccctt gatgatttcg
    10741 acctggacat gctgattaac tctagaagtt ccggatctcc gaaaaagaaa cgcaaagttg
    10801 gtagccagta cctgcccgac accgacgacc ggcaccggat cgaggaaaag cggaagcgga
    10861 cctacgagac attcaagagc atcatgaaga agtccccctt cagcggcccc accgacccta
    10921 gacctccacc tagaagaatc gccgtgccca gcagatccag cgccagcgtg ccaaaacctg
    10981 ccccccagcc ttaccccttc accagcagcc tgagcaccat caactacgac gagttcccta
    11041 ccatggtgtt ccccagcggc cagatctctc aggcctctgc tctggctcca gcccctcctc
    11101 aggtgctgcc tcaggctcct gctcctgcac cagctccagc catggtgtct gcactggctc
    11161 aggcaccagc acccgtgcct gtgctggctc ctggacctcc acaggctgtg gctccaccag
    11221 cccctaaacc tacacaggcc ggcgagggca cactgtctga agctctgctg cagctgcagt
    11281 tcgacgacga ggatctggga gccctgctgg gaaacagcac cgatcctgcc gtgttcaccg
    11341 acctggccag cgtggacaac agcgagttcc agcagctgct gaaccagggc atccctgtgg
    11401 cccctcacac caccgagccc atgctgatgg aataccccga ggccatcacc cggctcgtga
    11461 caggcgctca gaggcctcct gatccagctc ctgcccctct gggagcacca ggcctgccta
    11521 atggactgct gtctggcgac gaggacttca gctctatcgc cgacatggac ttctccgcac
    11581 tgctgggtag cggatcggga tctcgggatt ccagggaagg gatgtttttg ccgaagcctg
    11641 aggccggctc cgctattagt gacgtgtttg agggccgcga ggtgtgccag ccaaaacgaa
    11701 tccggccatt tcatcctcca ggaagtccat gggccaaccg cccactcccc gccagcctcg
    11761 caccaacacc aaccggtcca gtacatgagc cagtcgggtc actgaccccg gcaccagtcc
    11821 ctcagccact ggatccagcg cccgcagtga ctcccgaggc cagtcacctg ttggaggatc
    11881 ccgatgaaga gacgagccag gctgtcaaag cccttcggga gatggccgat actgtgattc
    11941 cccagaagga agaggctgca atctgtggcc aaatggacct ttcccatccg cccccaaggg
    12001 gccatctgga tgagctgaca accacacttg agtccatgac cgaggatctg aacctggact
    12061 cacccctgac cccggaattg aacgagattc tggatacctt cctgaacgac gagtgcctct
    12121 tgcatgccat gcatatcagc acaggactgt ccatcttcga cacatctctg ttttgaattg
    12181 caaagaacta gagccgcggg atctttgtga aggaacctta cttctgtggt gtgacataat
    12241 tggacaaact acctacagag atttaaagct ctaaggtaaa tataaaattt ttaagtgtat
    12301 aatgtgttaa actactgatt ctaattgttt gtgtatttta gattccaacc tatggaactg
    12361 atgaatggga gcagtggtgg aatgccttta atgaggaaaa cctgttttgc tcagaagaaa
    12421 tgccatctag tgatgatgag gctactgctg actctcaaca ttctactcct ccaaaaaaga
    12481 agagaaaggt agaagacccc aaggactttc cttcagaatt gctaagtttt ttgagtcatg
    12541 ctgtgtttag taatagaact cttgcttgct ttgctattta caccacaaag gaaaaagctg
    12601 cactgctata caagaaaatt atggaaaaat atttgatgta tagtgccttg actagagatc
    12661 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
    12721 cccctgaacc tgaaacataa aatgaatgga attgttgttg ttaacttgtt tattgcagct
    12781 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
    12841 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggttccag
    12901 agctctggcc acgtaataag tgtgcgttga atttattcgc aaaaacattg catattttcg
    12961 gcaaagtaaa attttgttgc ataccttatc aaaaaataag tgctgcatac tttttagaga
    13021 aaccaaataa ttttttattg catacccgtt tttaataaaa tacattgcat accctctttt
    13081 aataaaaaat attgcatact ttgacgaaac aaattttcgt tgcataccca ataaaagatt
    13141 attatattgc atacccgttt ttaataaaat acattgcata ccctctttta ataaaaaata
    13201 ttgcatacgt tgacgaaaca aattttcgtt gcatacccaa taaaagatta ttatattgca
    13261 taccttttct tgccatacca tttagccgat caattgtgct cggcaacagt atatttgtgg
    13321 tgtgccaacc aacaacgagc tccagctttt gttcccttta gtgagggtta atttcgagct
    13381 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
    13441 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
    13501 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
    13561 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
    13621 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
    13681 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
    13741 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
    13801 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
    13861 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
    13921 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
    13981 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
    14041 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
    14101 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
    14161 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
    14221 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
    14281 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
    14341 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
    14401 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
    14461 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
    14521 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
    14581 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
    14641 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
    14701 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
    14761 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
    14821 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
    14881 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
    14941 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
    15001 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
    15061 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
    15121 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
    15181 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
    15241 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
    15301 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
    15361 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
    15421 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
    15481 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
//
