LOCUS       sequence-206349-       12791 bp    DNA     circular SYN 15-APR-2025
DEFINITION  dCas9 fused to BFP and EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12791)
  AUTHORS   Lin L, Liu Y, Xu F, Huang J, Daugaard TF, Petersen TS, Hansen B, Ye 
            L, Zhou Q, Fang F, Yang L, Li S, Floe L, Jensen KT, Shrock E, Chen 
            F, Yang H, Wang J, Liu X, Xu X, Bolund L, Nielsen AL, Luo Y
  TITLE     Genome-wide determination of on-target and off-target 
            characteristics for RNA-guided DNA methylation by dCas9 
            methyltransferases.
  JOURNAL   Gigascience. 2018 Mar 1;7(3):1-19. doi: 10.1093/gigascience/giy011.
  PUBMED    29635374
REFERENCE   2  (bases 1 to 12791)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Gigascience"; date: "2018-03-1- 1"; volume: "7"; issue: "3"; pages:
            "1-19"
COMMENT     Sequence Label: pCCL-PGK-SPdCas9-BFP-EGFP
FEATURES             Location/Qualifiers
     source          1..12791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        82..461
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        462..665
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     616..636
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             680..860
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    904..1029
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1522..1755
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1939..1983
                     /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    2250..2367
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2406..2905
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2441..2464)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2838..2857
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2964..2973
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2970..2996,2997..2999,3000..5486,5487..5489,
                     5490..7073)
                     /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             2970..7073
                     /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             7077..7103
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7083..7109
                     /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             7122..7142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7149..7169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7209..7904
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     CDS             7956..8669
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             7956..8666
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(8256..8275)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8603..8624
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    8718..9306
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8771..8791)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9189..9200)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9395..9628
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(9657..9675)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(9658..9675)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(9933..9952)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9934..9952)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9962..9979)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(9962..9978)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9971..9993)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      10123..10578
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10210..10229)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10420..10441
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        10607..10711
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(10712..10780,10781..11572)
                     /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(10930..10949)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      11743..12331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12232..12251
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     12485..12502
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    12619..12640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(12655..12660,12661..12678,12679..12685)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12693..12709
                     /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)."
     primer_bind     12698..12720
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     12717..12733
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12717..12733
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     12752..12772
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        12754..12772
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat
       61 gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta
      121 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      181 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      241 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      301 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      361 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      421 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      481 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      541 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      601 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      661 gagctcgttt agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc
      721 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
      781 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
      841 agtgtggaaa atctctagca gtggcgcccg aacagggacc tgaaagcgaa agggaaacca
      901 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
      961 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     1021 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     1081 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     1141 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     1201 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     1261 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     1321 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     1381 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     1441 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     1501 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     1561 cactatgggc gcagcctcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     1621 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     1681 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     1741 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     1801 gccttggaat gctagttgga gtaataaatc tctggaacag attggaatca cacgacctgg
     1861 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     1921 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     1981 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     2041 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     2101 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     2161 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     2221 gtgaacggat ctcgacggta tcggttaact tttaaaagaa aaggggggat tggggggtac
     2281 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     2341 aaacaaatta caaaaattca aaattttatc gatcacgaga ctagcctcga cgatggtcga
     2401 gtaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     2461 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     2521 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     2581 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     2641 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     2701 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     2761 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     2821 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     2881 ctcttcctca tctccgggcc tttcgacctc tagcgggatc cggcgcgtct gcaggatatc
     2941 aagcttgcgg taccgcgggc ccggccacca tggacaagaa gtacagcatc ggcctggcca
     3001 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     3061 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggcgccc
     3121 tgctgttcga cagcggagaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     3181 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     3241 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     3301 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     3361 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     3421 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     3481 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     3541 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     3601 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     3661 gcgagaagaa gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca
     3721 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     3781 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     3841 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     3901 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     3961 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     4021 tcttcgacca gagcaagaac ggctacgccg gctacatcga tggcggagcc agccaggaag
     4081 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     4141 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     4201 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     4261 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     4321 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     4381 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgccagc gcccagagct
     4441 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     4501 acagcctgct gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg aaatacgtga
     4561 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaagcc atcgtggacc
     4621 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     4681 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     4741 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     4801 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     4861 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     4921 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     4981 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     5041 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     5101 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     5161 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     5221 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     5281 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     5341 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     5401 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     5461 accggctgtc cgactacgat gtggacgcta tcgtgcctca gagctttctg aaggacgact
     5521 ccatcgataa caaagtgctg actcggagcg acaagaaccg gggcaagagc gacaacgtgc
     5581 cctccgaaga ggtcgtgaag aagatgaaga actactggcg ccagctgctg aatgccaagc
     5641 tgattaccca gaggaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     5701 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     5761 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaacgac aaactgatcc
     5821 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     5881 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     5941 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     6001 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     6061 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     6121 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaacaggcg
     6181 agatcgtgtg ggataagggc cgggactttg ccaccgtgcg gaaagtgctg tctatgcccc
     6241 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     6301 tgcccaagag gaacagcgac aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     6361 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     6421 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     6481 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     6541 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     6601 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     6661 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     6721 ataatgagca gaaacagctg tttgtggaac agcacaaaca ctacctggac gagatcatcg
     6781 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaggtgc
     6841 tgagcgccta caacaagcac agagacaagc ctatcagaga gcaggccgag aatatcatcc
     6901 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     6961 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     7021 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacgcctatc
     7081 cctatgacgt gcccgattat gccagcctgg gcagcggctc ccccaagaaa aaacgcaagg
     7141 tggaagatcc taagaaaaag cggaaagtgg acggcattgg tagtgggagc aacggcagca
     7201 gcggatccag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
     7261 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     7321 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     7381 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
     7441 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
     7501 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     7561 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     7621 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     7681 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata
     7741 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     7801 tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg
     7861 ccagatactg cgacctccct agcaaactgg ggcacaagct taatggtgga ggaggttctg
     7921 gaggcggtgg aagtggtggc ggaggtagcg tcgacagcaa gggcgaggag ctgttcaccg
     7981 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     8041 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     8101 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     8161 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     8221 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     8281 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     8341 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     8401 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     8461 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     8521 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     8581 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     8641 ctctcggcat ggacgagctg tacaagtaat agatgcatgg ccggccttaa ttaaacgcgt
     8701 ctcgagggaa ttccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     8761 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     8821 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     8881 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     8941 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     9001 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     9061 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt
     9121 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     9181 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     9241 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     9301 ccccgcatcg ggaattcgag ctcggtacct ttaagaccaa tgacttacaa ggcagctgta
     9361 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     9421 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     9481 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     9541 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     9601 ttttagtcag tgtggaaaat ctctagcagc atctagctag aattaattcc gtgtattcta
     9661 tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt gtagccgcgt
     9721 tctaacgaca atatgtacaa gcctaattgt gtagcatctg gcttactgaa gcagacccta
     9781 tcatctctct cgtaaactgc cgtcagagtc ggtttggttg gacgaacctt ctgagtttct
     9841 ggtaacgccg tcccgcaccc ggaaatggtc agcgaaccaa tcagcagggt catcgctagc
     9901 ctaggctttt gcgtcgagac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     9961 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
    10021 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
    10081 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg
    10141 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
    10201 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
    10261 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
    10321 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
    10381 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
    10441 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
    10501 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
    10561 tattaacgtt tacaatttcc caggtggcac ttttcgggga aatgtgcgcg gaacccctat
    10621 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
    10681 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
    10741 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
    10801 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
    10861 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
    10921 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
    10981 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
    11041 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
    11101 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
    11161 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
    11221 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
    11281 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
    11341 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
    11401 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
    11461 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
    11521 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
    11581 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
    11641 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
    11701 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
    11761 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
    11821 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
    11881 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
    11941 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
    12001 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
    12061 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
    12121 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
    12181 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
    12241 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
    12301 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
    12361 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
    12421 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
    12481 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
    12541 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
    12601 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
    12661 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
    12721 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
    12781 gctggagctg c
//
