LOCUS       sequence-199744-       14167 bp    DNA     circular SYN 16-APR-2025
DEFINITION  Human expression vector containing SFFV promoter, dCas9 that is 
            fused to 2x NLS, tagBFP and a KRAB domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14167)
  AUTHORS   Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, 
            Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, 
            Weissman JS, Qi LS
  TITLE     CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in 
            Eukaryotes.
  JOURNAL   Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 
            10.1016/j.cell.2013.06.044.
  PUBMED    23849981
REFERENCE   2  (bases 1 to 14167)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2013 
            Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044."
COMMENT     Sequence Label: pHR-SFFV-dCas9-BFP-KRAB
FEATURES             Location/Qualifiers
     source          1..14167
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    668..793
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1290..1523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1708..1752
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2037..2154
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2307..2714
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2783..2792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2789..2815,2816..2818,2819..5305,5306..5308,
                     5309..6892)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2789..6892
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6896..6922
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             6902..6928
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             6941..6961
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6968..6988
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7028..7723
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             7769..7954
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     misc_feature    8018..8606
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8071..8091)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8489..8500)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(8609..8625)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8610..8626)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             8716..8949
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(8974..8992)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(8975..8992)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(9282..9301)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(9515..9534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9634..9656
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9694..9712)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9780..9884
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9885..9953,9954..10745)
                     /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     complement(10103..10122)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10916..11504
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     11405..11424
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     11658..11675
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(11745..11765)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11750..12079
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11930..12065
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          13213..13278
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             13408..13428
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    13853..13987
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13890..13909)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13944..13963
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             join(14155..14167,1..621)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 tcactcccaa agaagacaag atatccttga tctgtggatc taccacacac aaggctactt
       61 ccctgattag cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg
      121 gtgctacaag ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa
      181 caccagcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt
      241 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga
      301 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc
      361 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc
      421 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      481 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      541 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      601 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
      661 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
      721 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
      781 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
      841 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
      901 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
      961 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1021 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1081 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     1141 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1201 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1261 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1321 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1381 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1441 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1501 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1561 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1621 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1681 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1741 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1801 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1861 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     1921 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     1981 attcgattag tgaacggatc tcgacggtat cgccaaatgg cagtattcat ccacaatttt
     2041 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     2101 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     2161 tattacaggg acagcagaga tccagtttgg atcgataagc ttgatatcga attcctgcag
     2221 ccccgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca
     2281 cctgtaggtt tggcaagcta gctgcagtaa cgccattttg caaggcatgg aaaaatacca
     2341 aaccaagaat agagaagttc agatcaaggg cgggtacatg aaaatagcta acgttgggcc
     2401 aaacaggata tctgcggtga gcagtttcgg ccccggcccg gggccaagaa cagatggtca
     2461 ccgcagtttc ggccccggcc cgaggccaag aacagatggt ccccagatat ggcccaaccc
     2521 tcagcagttt cttaagaccc atcagatgtt tccaggctcc cccaaggacc tgaaatgacc
     2581 ctgcgcctta tttgaattaa ccaatcagcc tgcttctcgc ttctgttcgc gcgcttctgc
     2641 ttcccgagct ctataaaaga gctcacaacc cctcactcgg cgcgccagtc ctccgacaga
     2701 ctgagtcgcc cgggggggat ctggagctct cgagaattct cacgcgtctg caggatatca
     2761 agcttgcggt accgcgggcc cggccaccat ggacaagaag tacagcatcg gcctggccat
     2821 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     2881 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggcgccct
     2941 gctgttcgac agcggagaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     3001 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     3061 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     3121 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     3181 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     3241 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     3301 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     3361 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     3421 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     3481 cgagaagaag aatggcctgt tcggcaacct gattgccctg agcctgggcc tgacccccaa
     3541 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     3601 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     3661 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     3721 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     3781 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     3841 cttcgaccag agcaagaacg gctacgccgg ctacatcgat ggcggagcca gccaggaaga
     3901 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     3961 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     4021 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     4081 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     4141 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     4201 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgccagcg cccagagctt
     4261 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     4321 cagcctgctg tacgagtact tcaccgtgta caacgagctg accaaagtga aatacgtgac
     4381 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaagcca tcgtggacct
     4441 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     4501 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     4561 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     4621 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     4681 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     4741 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     4801 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     4861 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     4921 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     4981 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     5041 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     5101 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     5161 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     5221 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     5281 ccggctgtcc gactacgatg tggacgctat cgtgcctcag agctttctga aggacgactc
     5341 catcgataac aaagtgctga ctcggagcga caagaaccgg ggcaagagcg acaacgtgcc
     5401 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgc cagctgctga atgccaagct
     5461 gattacccag aggaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     5521 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     5581 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaacgaca aactgatccg
     5641 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     5701 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     5761 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     5821 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     5881 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     5941 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaacaggcga
     6001 gatcgtgtgg gataagggcc gggactttgc caccgtgcgg aaagtgctgt ctatgcccca
     6061 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     6121 gcccaagagg aacagcgaca agctgatcgc cagaaagaag gactgggacc ctaagaagta
     6181 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     6241 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     6301 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     6361 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     6421 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     6481 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     6541 taatgagcag aaacagctgt ttgtggaaca gcacaaacac tacctggacg agatcatcga
     6601 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaggtgct
     6661 gagcgcctac aacaagcaca gagacaagcc tatcagagag caggccgaga atatcatcca
     6721 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     6781 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     6841 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acgcctatcc
     6901 ctatgacgtg cccgattatg ccagcctggg cagcggctcc cccaagaaaa aacgcaaggt
     6961 ggaagatcct aagaaaaagc ggaaagtgga cggcattggt agtgggagca acggcagcag
     7021 cggatccagc gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt
     7081 ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca
     7141 gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc
     7201 tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt
     7261 cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg
     7321 gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt
     7381 caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg
     7441 ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga
     7501 catggccctg aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag
     7561 atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact
     7621 ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc
     7681 cagatactgc gacctcccta gcaaactggg gcacaagctt aatggcggtg gcggagggat
     7741 ggatgctaag tcactaactg cctggtcccg gacactggtg accttcaagg atgtatttgt
     7801 ggacttcacc agggaggagt ggaagctgct ggacactgct cagcagatcg tgtacagaaa
     7861 tgtgatgctg gagaactata agaacctggt ttccttgggt tatcagctta ctaagccaga
     7921 tgtgatcctc cggttggaga agggagaaga gccctagtaa ggccgcgact ctagagtcga
     7981 cctgcaggca tgcaagcttg atatcaagct tatcgataat caacctctgg attacaaaat
     8041 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     8101 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     8161 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     8221 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     8281 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     8341 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     8401 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct
     8461 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     8521 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     8581 gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgaggga attaattcga
     8641 gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     8701 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     8761 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     8821 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     8881 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     8941 tctctagcag catctagaat taattccgtg tattctatag tgtcacctaa atcgtatgtg
     9001 tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata tgtacaagcc
     9061 taattgtgta gcatctggct tactgaagca gaccctatca tctctctcgt aaactgccgt
     9121 cagagtcggt ttggttggac gaaccttctg agtttctggt aacgccgtcc cgcacccgga
     9181 aatggtcagc gaaccaatca gcagggtcat cgctagccag atcctctacg ccggacgcat
     9241 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac
     9301 cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat
     9361 ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
     9421 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc
     9481 gcataaggga gagcgtcgaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     9541 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     9601 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     9661 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     9721 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     9781 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     9841 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     9901 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     9961 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
    10021 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
    10081 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
    10141 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
    10201 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
    10261 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
    10321 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
    10381 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
    10441 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
    10501 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
    10561 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
    10621 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
    10681 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
    10741 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
    10801 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
    10861 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
    10921 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
    10981 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
    11041 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
    11101 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
    11161 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
    11221 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
    11281 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
    11341 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
    11401 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
    11461 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
    11521 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
    11581 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
    11641 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
    11701 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgtggaatg tgtgtcagtt
    11761 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
    11821 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
    11881 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
    11941 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
    12001 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
    12061 aggcctaggc ttttgcaaaa agcttggaca caagacaggc ttgcgagata tgtttgagaa
    12121 taccacttta tcccgcgtca gggagaggca gtgcgtaaaa agacgcggac tcatgtgaaa
    12181 tactggtttt tagtgcgcca gatctctata atctcgcgca acctattttc ccctcgaaca
    12241 ctttttaagc cgtagataaa caggctggga cacttcacat gagcgaaaaa tacatcgtca
    12301 cctgggacat gttgcagatc catgcacgta aactcgcaag ccgactgatg ccttctgaac
    12361 aatggaaagg cattattgcc gtaagccgtg gcggtctgta ccgggtgcgt tactggcgcg
    12421 tgaactgggt attcgtcatg tcgataccgt ttgtatttcc agctacgatc acgacaacca
    12481 gcgcgagctt aaagtgctga aacgcgcaga aggcgatggc gaaggcttca tcgttattga
    12541 tgacctggtg gataccggtg gtactgcggt tgcgattcgt gaaatgtatc caaaagcgca
    12601 ctttgtcacc atcttcgcaa aaccggctgg tcgtccgctg gttgatgact atgttgttga
    12661 tatcccgcaa gatacctgga ttgaacagcc gtgggatatg ggcgtcgtat tcgtcccgcc
    12721 aatctccggt cgctaatctt ttcaacgcct ggcactgccg ggcgttgttc tttttaactt
    12781 caggcgggtt acaatagttt ccagtaagta ttctggaggc tgcatccatg acacaggcaa
    12841 acctgagcga aaccctgttc aaaccccgct ttaaacatcc tgaaacctcg acgctagtcc
    12901 gccgctttaa tcacggcgca caaccgcctg tgcagtcggc ccttgatggt aaaaccatcc
    12961 ctcactggta tcgcatgatt aaccgtctga tgtggatctg gcgcggcatt gacccacgcg
    13021 aaatcctcga cgtccaggca cgtattgtga tgagcgatgc cgaacgtacc gacgatgatt
    13081 tatacgatac ggtgattggc taccgtggcg gcaactggat ttatgagtgg gccccggatc
    13141 tttgtgaagg aaccttactt ctgtggtgtg acataattgg acaaactacc tacagagatt
    13201 taaagctcta aggtaaatat aaaattttta agtgtataat gtgttaaact actgattcta
    13261 attgtttgtg tattttagat tccaacctat ggaactgatg aatgggagca gtggtggaat
    13321 gcctttaatg aggaaaacct gttttgctca gaagaaatgc catctagtga tgatgaggct
    13381 actgctgact ctcaacattc tactcctcca aaaaagaaga gaaaggtaga agaccccaag
    13441 gactttcctt cagaattgct aagttttttg agtcatgctg tgtttagtaa tagaactctt
    13501 gcttgctttg ctatttacac cacaaaggaa aaagctgcac tgctatacaa gaaaattatg
    13561 gaaaaatatt ctgtaacctt tataagtagg cataacagtt ataatcataa catactgttt
    13621 tttcttactc cacacaggca tagagtgtct gctattaata actatgctca aaaattgtgt
    13681 acctttagct ttttaatttg taaaggggtt aataaggaat atttgatgta tagtgccttg
    13741 actagagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
    13801 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
    13861 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
    13921 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
    13981 ctggatcaac tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc
    14041 tattaccact gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt
    14101 attttcattt taaagaaatt gtatttgtta aatatgtact acaaacttag tagttggaag
    14161 ggctaat
//
