LOCUS       sequence-374755-       15713 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 15713)
  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 15713)
  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-P2A-BFP
FEATURES             Location/Qualifiers
     source          1..15713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(62..82)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        67..396
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      247..382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          1530..1595
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             1725..1745
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2170..2304
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2207..2226)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2261..2280
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             2472..3105
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3152..3277
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    3774..4007
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             4192..4236
                     /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    4521..4638
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        5370..6547
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          5601..6539
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     6496..6516
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(6904..6928)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             6948..6968
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7017..7026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(7023..7049,7050..7052,7053..9539,9540..9542,
                     9543..11126)
                     /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             7023..11126
                     /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             11130..11156
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             11136..11162
                     /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             11175..11195
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11202..11222
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             11232..11288
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             11289..11996
                     /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    12048..12636
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(12101..12121)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(12519..12530)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12639..12655)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12640..12656)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             12746..12979
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(13004..13022)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(13005..13022)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(13312..13331)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(13545..13564)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     13664..13686
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(13724..13742)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        13810..13914
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(13915..13983,13984..14775)
                     /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(14133..14152)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      14946..15534
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     15435..15454
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     15688..15705
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
       61 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
      121 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
      181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
      241 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
      301 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
      361 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttggacacaa gacaggcttg
      421 cgagatatgt ttgagaatac cactttatcc cgcgtcaggg agaggcagtg cgtaaaaaga
      481 cgcggactca tgtgaaatac tggtttttag tgcgccagat ctctataatc tcgcgcaacc
      541 tattttcccc tcgaacactt tttaagccgt agataaacag gctgggacac ttcacatgag
      601 cgaaaaatac atcgtcacct gggacatgtt gcagatccat gcacgtaaac tcgcaagccg
      661 actgatgcct tctgaacaat ggaaaggcat tattgccgta agccgtggcg gtctgtaccg
      721 ggtgcgttac tggcgcgtga actgggtatt cgtcatgtcg ataccgtttg tatttccagc
      781 tacgatcacg acaaccagcg cgagcttaaa gtgctgaaac gcgcagaagg cgatggcgaa
      841 ggcttcatcg ttattgatga cctggtggat accggtggta ctgcggttgc gattcgtgaa
      901 atgtatccaa aagcgcactt tgtcaccatc ttcgcaaaac cggctggtcg tccgctggtt
      961 gatgactatg ttgttgatat cccgcaagat acctggattg aacagccgtg ggatatgggc
     1021 gtcgtattcg tcccgccaat ctccggtcgc taatcttttc aacgcctggc actgccgggc
     1081 gttgttcttt ttaacttcag gcgggttaca atagtttcca gtaagtattc tggaggctgc
     1141 atccatgaca caggcaaacc tgagcgaaac cctgttcaaa ccccgcttta aacatcctga
     1201 aacctcgacg ctagtccgcc gctttaatca cggcgcacaa ccgcctgtgc agtcggccct
     1261 tgatggtaaa accatccctc actggtatcg catgattaac cgtctgatgt ggatctggcg
     1321 cggcattgac ccacgcgaaa tcctcgacgt ccaggcacgt attgtgatga gcgatgccga
     1381 acgtaccgac gatgatttat acgatacggt gattggctac cgtggcggca actggattta
     1441 tgagtgggcc ccggatcttt gtgaaggaac cttacttctg tggtgtgaca taattggaca
     1501 aactacctac agagatttaa agctctaagg taaatataaa atttttaagt gtataatgtg
     1561 ttaaactact gattctaatt gtttgtgtat tttagattcc aacctatgga actgatgaat
     1621 gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt ttgctcagaa gaaatgccat
     1681 ctagtgatga tgaggctact gctgactctc aacattctac tcctccaaaa aagaagagaa
     1741 aggtagaaga ccccaaggac tttccttcag aattgctaag ttttttgagt catgctgtgt
     1801 ttagtaatag aactcttgct tgctttgcta tttacaccac aaaggaaaaa gctgcactgc
     1861 tatacaagaa aattatggaa aaatattctg taacctttat aagtaggcat aacagttata
     1921 atcataacat actgtttttt cttactccac acaggcatag agtgtctgct attaataact
     1981 atgctcaaaa attgtgtacc tttagctttt taatttgtaa aggggttaat aaggaatatt
     2041 tgatgtatag tgccttgact agagatcata atcagccata ccacatttgt agaggtttta
     2101 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2161 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2221 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2281 aatgtatctt atcatgtctg gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa
     2341 cttcccaccc cataccctat taccactgcc aattacctgt ggtttcattt actctaaacc
     2401 tgtgattcct ctgaattatt ttcattttaa agaaattgta tttgttaaat atgtactaca
     2461 aacttagtag ttggaagggc taattcactc ccaaagaaga caagatatcc ttgatctgtg
     2521 gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt
     2581 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt
     2641 agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat
     2701 ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca
     2761 cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa
     2821 gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg
     2881 agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag
     2941 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     3001 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     3061 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
     3121 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
     3181 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
     3241 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
     3301 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
     3361 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
     3421 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
     3481 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
     3541 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
     3601 caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     3661 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     3721 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     3781 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     3841 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     3901 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     3961 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     4021 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     4081 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     4141 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     4201 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     4261 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     4321 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     4381 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     4441 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgccaa
     4501 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     4561 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     4621 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt ttggatcgat
     4681 aagcttgata tcgaattcgg gaggtggtcc ctgcagttac gccaatgata acccccgcca
     4741 gaaaaatctt agtagccttc cctttttgtt ttccgtgccc caactcggcg gattgactcg
     4801 gccccttccg gaaacacccg aatcaacttc tagtcaaatt attgttcacg ccgcaatgac
     4861 ccacccctgg cccgcgtctg tggaactgac ccctggtgta caggagagtt cgctgctgaa
     4921 agtggtccca aaggggtact agtttttaag ctcccaactc cccctccccc agcgtctgga
     4981 ggattccaca ccctcgcacc gcaggggcga ggaagtgggc ggagtccggt tttggcgcca
     5041 gccgctgagg ctgccaagca gaaaagccac cgctgaggag actccggtca ctgtcctcgc
     5101 cccgcctccc ccttccctcc ccttggggac caccgggcgc cacgccgcga acggtaagtg
     5161 ccgcggtcgt cggcgcctcc gccctccccc tagggcccca attcccagcg ggcgcggcgc
     5221 gcggcccctc cccccgccgg gcgcgcgccc gctgccccgc ccttcgtggc cgcccggcgt
     5281 gggcggtgcc acccctcccc ccggcggccc cgcgcgcagc tcccggctcc ctcccccttc
     5341 ggatgtggct tgagctgtag gcgcggaggg gctccggtgc ccgtcagtgg gcagagcgca
     5401 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     5461 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     5521 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     5581 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
     5641 cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
     5701 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
     5761 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
     5821 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
     5881 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
     5941 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
     6001 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
     6061 ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct
     6121 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
     6181 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
     6241 cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
     6301 agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt
     6361 taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
     6421 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     6481 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
     6541 tgtcgtggaa ttctcacgcg tctgcaggat atcaagcttg cggtaccgcg ggcccggcca
     6601 ccatgaacaa ttcccaggga agagtgacct tcgaggatgt cactgtgaac ttcacccagg
     6661 gggagtggca gcggctgaat cccgaacaga gaaacttgta cagggatgtg atgctggaga
     6721 attacagcaa ccttgtctct gtgggacaag gggaaaccac caaacccgat gtgatcttga
     6781 ggttggaaca aggaaaggag ccatggttgg aggaagagga agtgctggga agtggccgtg
     6841 cagaaaaaaa tggggacatt ggagggcaga tttggaagcc aaaggatgtg aaagagagtc
     6901 tctacccaac tttcttgtac aaagtggttg gtggatctgg aggttctccc aagaagaagc
     6961 gcaaggtggg acgcgtctgc aggatatcaa gcttgcggta ccgcgggccc gggatcgcca
     7021 ccatggacaa gaagtacagc atcggcctgg ccatcggcac caactctgtg ggctgggccg
     7081 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
     7141 ggcacagcat caagaagaac ctgatcggcg ccctgctgtt cgacagcgga gaaacagccg
     7201 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
     7261 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
     7321 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
     7381 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
     7441 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     7501 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     7561 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     7621 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     7681 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggca
     7741 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     7801 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     7861 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     7921 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     7981 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     8041 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     8101 ccggctacat cgatggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     8161 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     8221 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     8281 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     8341 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     8401 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     8461 aagtggtgga caagggcgcc agcgcccaga gcttcatcga gcggatgacc aacttcgata
     8521 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     8581 tgtacaacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     8641 tgagcggcga gcagaaaaaa gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     8701 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     8761 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     8821 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     8881 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     8941 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     9001 gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc
     9061 tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg
     9121 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     9181 tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga
     9241 cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg
     9301 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga
     9361 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     9421 ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc
     9481 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacg
     9541 ctatcgtgcc tcagagcttt ctgaaggacg actccatcga taacaaagtg ctgactcgga
     9601 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     9661 agaactactg gcgccagctg ctgaatgcca agctgattac ccagaggaag ttcgacaatc
     9721 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     9781 agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga
     9841 acactaagta cgacgagaac gacaaactga tccgggaagt gaaagtgatc accctgaagt
     9901 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     9961 actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa
    10021 agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga
    10081 agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca
    10141 gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc
    10201 ggcctctgat cgagacaaac ggcgaaacag gcgagatcgt gtgggataag ggccgggact
    10261 ttgccaccgt gcggaaagtg ctgtctatgc cccaagtgaa tatcgtgaaa aagaccgagg
    10321 tgcagacagg cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gacaagctga
    10381 tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg
    10441 cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg
    10501 tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg
    10561 actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta
    10621 agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac
    10681 tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca
    10741 gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg
    10801 aacagcacaa acactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag
    10861 tgatcctggc cgacgctaat ctggacaagg tgctgagcgc ctacaacaag cacagagaca
    10921 agcctatcag agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag
    10981 cccctgccgc cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca
    11041 aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga
    11101 tcgacctgtc tcagctggga ggcgacgcct atccctatga cgtgcccgat tatgccagcc
    11161 tgggcagcgg ctcccccaag aaaaaacgca aggtggaaga tcctaagaaa aagcggaaag
    11221 tgagcggccg cgctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc
    11281 ctggacctgt gtctaagggc gaagagctga ttaaggagaa catgcacatg aagctgtaca
    11341 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
    11401 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
    11461 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
    11521 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
    11581 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
    11641 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
    11701 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
    11761 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacgcca
    11821 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
    11881 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
    11941 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattaat
    12001 aagcgatcgc cctgcaggca tgcaagcttg atatcaagct tatcgataat caacctctgg
    12061 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
    12121 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
    12181 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
    12241 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
    12301 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
    12361 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
    12421 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca
    12481 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
    12541 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
    12601 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga cctcgaggga
    12661 attaattcga gctcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
    12721 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
    12781 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
    12841 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
    12901 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
    12961 gtgtggaaaa tctctagcag catctagaat taattccgtg tattctatag tgtcacctaa
    13021 atcgtatgtg tatgatacat aaggttatgt attaattgta gccgcgttct aacgacaata
    13081 tgtacaagcc taattgtgta gcatctggct tactgaagca gaccctatca tctctctcgt
    13141 aaactgccgt cagagtcggt ttggttggac gaaccttctg agtttctggt aacgccgtcc
    13201 cgcacccgga aatggtcagc gaaccaatca gcagggtcat cgctagccag atcctctacg
    13261 ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
    13321 ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc gcttgtttcg
    13381 gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg
    13441 caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
    13501 tgcaggagtc gcataaggga gagcgtcgaa tggtgcactc tcagtacaat ctgctctgat
    13561 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
    13621 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
    13681 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
    13741 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
    13801 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
    13861 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
    13921 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
    13981 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
    14041 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
    14101 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
    14161 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
    14221 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
    14281 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
    14341 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
    14401 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
    14461 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
    14521 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
    14581 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
    14641 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
    14701 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
    14761 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
    14821 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
    14881 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
    14941 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
    15001 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
    15061 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
    15121 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
    15181 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
    15241 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    15301 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    15361 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    15421 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    15481 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    15541 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
    15601 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
    15661 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc gga
//
