LOCUS       sequence-374758-       15704 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 with an N-term Zim3 KRAB-NLS fusion, a C-term HA-2xNLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15704)
  AUTHORS   Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, 
            Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, 
            Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M
  TITLE     Maximizing CRISPRi efficacy and accessibility with dual-sgRNA 
            libraries and optimal effectors.
  JOURNAL   eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.
  PUBMED    36576240
REFERENCE   2  (bases 1 to 15704)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "eLife."; 
            date: "2022-12-28"; volume: "11:e81856. doi"; pages: " 
            10.7554/eLife.81856"
COMMENT     Sequence Label: pHR-UCOE-EF1a-Zim3-dCas9-BFP
FEATURES             Location/Qualifiers
     source          1..15704
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     120..137
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(207..227)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        212..541
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      392..527
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1675..1740
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1870..1890
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2315..2449
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2352..2371)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2406..2425
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2617..3250
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3297..3422
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3919..4152
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4337..4381
                     /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    4666..4783
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5515..6692
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5746..6684
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6641..6661
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(7049..7073)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             7093..7113
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7162..7171
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7168..7194,7195..7197,7198..9684,9685..9687,
                     9688..11271)
                     /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             7168..11271
                     /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             11275..11301
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             11281..11307
                     /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             11320..11340
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11347..11367
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11425..12132
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     misc_feature    12184..12772
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12237..12257)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12655..12666)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12775..12791)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12776..12792)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12882..13115
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(13140..13158)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(13141..13158)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(13448..13467)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(13681..13700)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13800..13822
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13860..13878)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13946..14050
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(14051..14119,14120..14911)
                     /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(14269..14288)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      15082..15670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     15571..15590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
       61 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      121 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
      181 gttggccgat tcattaatgc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
      241 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
      301 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      361 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
      421 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
      481 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
      541 aaagcttgga cacaagacag gcttgcgaga tatgtttgag aataccactt tatcccgcgt
      601 cagggagagg cagtgcgtaa aaagacgcgg actcatgtga aatactggtt tttagtgcgc
      661 cagatctcta taatctcgcg caacctattt tcccctcgaa cactttttaa gccgtagata
      721 aacaggctgg gacacttcac atgagcgaaa aatacatcgt cacctgggac atgttgcaga
      781 tccatgcacg taaactcgca agccgactga tgccttctga acaatggaaa ggcattattg
      841 ccgtaagccg tggcggtctg taccgggtgc gttactggcg cgtgaactgg gtattcgtca
      901 tgtcgatacc gtttgtattt ccagctacga tcacgacaac cagcgcgagc ttaaagtgct
      961 gaaacgcgca gaaggcgatg gcgaaggctt catcgttatt gatgacctgg tggataccgg
     1021 tggtactgcg gttgcgattc gtgaaatgta tccaaaagcg cactttgtca ccatcttcgc
     1081 aaaaccggct ggtcgtccgc tggttgatga ctatgttgtt gatatcccgc aagatacctg
     1141 gattgaacag ccgtgggata tgggcgtcgt attcgtcccg ccaatctccg gtcgctaatc
     1201 ttttcaacgc ctggcactgc cgggcgttgt tctttttaac ttcaggcggg ttacaatagt
     1261 ttccagtaag tattctggag gctgcatcca tgacacaggc aaacctgagc gaaaccctgt
     1321 tcaaaccccg ctttaaacat cctgaaacct cgacgctagt ccgccgcttt aatcacggcg
     1381 cacaaccgcc tgtgcagtcg gcccttgatg gtaaaaccat ccctcactgg tatcgcatga
     1441 ttaaccgtct gatgtggatc tggcgcggca ttgacccacg cgaaatcctc gacgtccagg
     1501 cacgtattgt gatgagcgat gccgaacgta ccgacgatga tttatacgat acggtgattg
     1561 gctaccgtgg cggcaactgg atttatgagt gggccccgga tctttgtgaa ggaaccttac
     1621 ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat
     1681 ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag
     1741 attccaacct atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac
     1801 ctgttttgct cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat
     1861 tctactcctc caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg
     1921 ctaagttttt tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac
     1981 accacaaagg aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc
     2041 tttataagta ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg
     2101 catagagtgt ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt
     2161 tgtaaagggg ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag
     2221 ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa
     2281 cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg
     2341 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     2401 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatca actggataac
     2461 tcaagctaac caaaatcatc ccaaacttcc caccccatac cctattacca ctgccaatta
     2521 cctgtggttt catttactct aaacctgtga ttcctctgaa ttattttcat tttaaagaaa
     2581 ttgtatttgt taaatatgta ctacaaactt agtagttgga agggctaatt cactcccaaa
     2641 gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc cctgattagc
     2701 agaactacac accagggcca ggggtcagat atccactgac ctttggatgg tgctacaagc
     2761 tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac accagcttgt
     2821 tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta gagtggaggt
     2881 ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag tacttcaaga
     2941 actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca gggaggcgtg
     3001 gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca gctgcttttt
     3061 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     3121 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     3181 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     3241 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     3301 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     3361 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     3421 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     3481 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     3541 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     3601 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     3661 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     3721 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag
     3781 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     3841 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     3901 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     3961 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     4021 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     4081 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     4141 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     4201 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     4261 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     4321 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     4381 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     4441 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     4501 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     4561 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     4621 gaacggatct cgacggtatc gccaaatggc agtattcatc cacaatttta aaagaaaagg
     4681 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     4741 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     4801 cagcagagat ccagtttgga tcgataagct tgatatcgaa ttcgggaggt ggtccctgca
     4861 gttacgccaa tgataacccc cgccagaaaa atcttagtag ccttcccttt ttgttttccg
     4921 tgccccaact cggcggattg actcggcccc ttccggaaac acccgaatca acttctagtc
     4981 aaattattgt tcacgccgca atgacccacc cctggcccgc gtctgtggaa ctgacccctg
     5041 gtgtacagga gagttcgctg ctgaaagtgg tcccaaaggg gtactagttt ttaagctccc
     5101 aactccccct cccccagcgt ctggaggatt ccacaccctc gcaccgcagg ggcgaggaag
     5161 tgggcggagt ccggttttgg cgccagccgc tgaggctgcc aagcagaaaa gccaccgctg
     5221 aggagactcc ggtcactgtc ctcgccccgc ctcccccttc cctccccttg gggaccaccg
     5281 ggcgccacgc cgcgaacggt aagtgccgcg gtcgtcggcg cctccgccct ccccctaggg
     5341 ccccaattcc cagcgggcgc ggcgcgcggc ccctcccccc gccgggcgcg cgcccgctgc
     5401 cccgcccttc gtggccgccc ggcgtgggcg gtgccacccc tccccccggc ggccccgcgc
     5461 gcagctcccg gctccctccc ccttcggatg tggcttgagc tgtaggcgcg gaggggctcc
     5521 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
     5581 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
     5641 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
     5701 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
     5761 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
     5821 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
     5881 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
     5941 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
     6001 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     6061 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     6121 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     6181 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     6241 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     6301 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     6361 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     6421 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     6481 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     6541 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     6601 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     6661 caaagttttt ttcttccatt tcaggtgtcg tggaattctc acgcgtctgc aggatatcaa
     6721 gcttgcggta ccgcgggccc ggccaccatg aacaattccc agggaagagt gaccttcgag
     6781 gatgtcactg tgaacttcac ccagggggag tggcagcggc tgaatcccga acagagaaac
     6841 ttgtacaggg atgtgatgct ggagaattac agcaaccttg tctctgtggg acaaggggaa
     6901 accaccaaac ccgatgtgat cttgaggttg gaacaaggaa aggagccatg gttggaggaa
     6961 gaggaagtgc tgggaagtgg ccgtgcagaa aaaaatgggg acattggagg gcagatttgg
     7021 aagccaaagg atgtgaaaga gagtctctac ccaactttct tgtacaaagt ggttggtgga
     7081 tctggaggtt ctcccaagaa gaagcgcaag gtgggacgcg tctgcaggat atcaagcttg
     7141 cggtaccgcg ggcccgggat cgccaccatg gacaagaagt acagcatcgg cctggccatc
     7201 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     7261 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggcgccctg
     7321 ctgttcgaca gcggagaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     7381 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     7441 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     7501 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     7561 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     7621 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     7681 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     7741 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     7801 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     7861 gagaagaaga atggcctgtt cggcaacctg attgccctga gcctgggcct gacccccaac
     7921 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     7981 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     8041 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     8101 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     8161 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     8221 ttcgaccaga gcaagaacgg ctacgccggc tacatcgatg gcggagccag ccaggaagag
     8281 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     8341 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     8401 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     8461 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     8521 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     8581 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc
     8641 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     8701 agcctgctgt acgagtactt caccgtgtac aacgagctga ccaaagtgaa atacgtgacc
     8761 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaagccat cgtggacctg
     8821 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     8881 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     8941 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     9001 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     9061 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     9121 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     9181 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     9241 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     9301 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     9361 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     9421 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     9481 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     9541 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     9601 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     9661 cggctgtccg actacgatgt ggacgctatc gtgcctcaga gctttctgaa ggacgactcc
     9721 atcgataaca aagtgctgac tcggagcgac aagaaccggg gcaagagcga caacgtgccc
     9781 tccgaagagg tcgtgaagaa gatgaagaac tactggcgcc agctgctgaa tgccaagctg
     9841 attacccaga ggaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     9901 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     9961 gcacagatcc tggactcccg gatgaacact aagtacgacg agaacgacaa actgatccgg
    10021 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
    10081 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
    10141 gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc
    10201 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
    10261 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
    10321 ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga aacaggcgag
    10381 atcgtgtggg ataagggccg ggactttgcc accgtgcgga aagtgctgtc tatgccccaa
    10441 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
    10501 cccaagagga acagcgacaa gctgatcgcc agaaagaagg actgggaccc taagaagtac
    10561 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
    10621 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
    10681 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
    10741 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
    10801 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
    10861 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
    10921 aatgagcaga aacagctgtt tgtggaacag cacaaacact acctggacga gatcatcgag
    10981 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaggtgctg
    11041 agcgcctaca acaagcacag agacaagcct atcagagagc aggccgagaa tatcatccac
    11101 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
    11161 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
    11221 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga cgcctatccc
    11281 tatgacgtgc ccgattatgc cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg
    11341 gaagatccta agaaaaagcg gaaagtgagc ggccgcagcg gaagtgagac cccaggtaca
    11401 tccgaatcag caacgcctga aagcgtgtct aagggcgaag agctgattaa ggagaacatg
    11461 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
    11521 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
    11581 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
    11641 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
    11701 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
    11761 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
    11821 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgagac gctgtacccc
    11881 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
    11941 ctgatcgcaa acgccaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
    12001 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc
    12061 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
    12121 cacaagctta attaataagc gatcgccctg caggcatgca agcttgatat caagcttatc
    12181 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
    12241 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
    12301 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
    12361 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
    12421 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
    12481 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
    12541 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
    12601 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
    12661 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
    12721 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
    12781 cgtcgacctc gagggaatta attcgagctc ggtaccttta agaccaatga cttacaaggc
    12841 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
    12901 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
    12961 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
    13021 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
    13081 cagacccttt tagtcagtgt ggaaaatctc tagcagcatc tagaattaat tccgtgtatt
    13141 ctatagtgtc acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg
    13201 cgttctaacg acaatatgta caagcctaat tgtgtagcat ctggcttact gaagcagacc
    13261 ctatcatctc tctcgtaaac tgccgtcaga gtcggtttgg ttggacgaac cttctgagtt
    13321 tctggtaacg ccgtcccgca cccggaaatg gtcagcgaac caatcagcag ggtcatcgct
    13381 agccagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
    13441 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
    13501 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
    13561 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
    13621 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaatggt gcactctcag
    13681 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
    13741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
    13801 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
    13861 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
    13921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
    13981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
    14041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
    14101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
    14161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
    14221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
    14281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
    14341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
    14401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
    14461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
    14521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
    14581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
    14641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
    14701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
    14761 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
    14821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
    14881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
    14941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
    15001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
    15061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
    15121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
    15181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
    15241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
    15301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
    15361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
    15421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
    15481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
    15541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
    15601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
    15661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctgg
//
