LOCUS       sequence-374972-       14969 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 14969)
  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 14969)
  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-SFFV-Zim3-dCas9-P2A-BFP
FEATURES             Location/Qualifiers
     source          1..14969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(23..41)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(24..41)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(331..350)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(564..583)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     683..705
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(743..761)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        829..933
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(934..1002,1003..1794)
                     /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(1152..1171)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1965..2553
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2454..2473
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2707..2724
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2794..2814)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2799..3128
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2979..3114
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4262..4327
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4457..4477
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4902..5036
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4939..4958)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4993..5012
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5204..5837
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5884..6009
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6506..6739
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6924..6968
                     /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    7253..7370
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8112..8519
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     protein_bind    complement(8895..8919)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             8939..8959
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      9008..9017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9014..9040,9041..9043,9044..11530,11531..11533,
                     11534..13117)
                     /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             9014..13117
                     /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             13121..13147
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             13127..13153
                     /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             13166..13186
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13193..13213
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13223..13279
                     /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             13280..13987
                     /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    14036..14624
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(14089..14109)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(14507..14518)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(14627..14643)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14628..14644)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             14734..14967
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 atctagaatt aattccgtgt attctatagt gtcacctaaa tcgtatgtgt atgatacata
       61 aggttatgta ttaattgtag ccgcgttcta acgacaatat gtacaagcct aattgtgtag
      121 catctggctt actgaagcag accctatcat ctctctcgta aactgccgtc agagtcggtt
      181 tggttggacg aaccttctga gtttctggta acgccgtccc gcacccggaa atggtcagcg
      241 aaccaatcag cagggtcatc gctagccaga tcctctacgc cggacgcatc gtggccggca
      301 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
      361 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
      421 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
      481 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
      541 agcgtcgaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
      601 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
      661 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
      721 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
      781 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
      841 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
      901 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
      961 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1021 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     1081 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     1141 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     1201 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     1261 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     1321 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     1381 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     1441 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     1501 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     1561 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     1621 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     1681 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     1741 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     1801 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     1861 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     1921 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1981 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2041 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2101 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2161 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2221 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2281 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2341 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2401 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2461 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2521 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2581 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2641 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2701 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     2761 cccgcgcgtt ggccgattca ttaatgcagc tgtggaatgt gtgtcagtta gggtgtggaa
     2821 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2881 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2941 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3001 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3061 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3121 tttgcaaaaa gcttggacac aagacaggct tgcgagatat gtttgagaat accactttat
     3181 cccgcgtcag ggagaggcag tgcgtaaaaa gacgcggact catgtgaaat actggttttt
     3241 agtgcgccag atctctataa tctcgcgcaa cctattttcc cctcgaacac tttttaagcc
     3301 gtagataaac aggctgggac acttcacatg agcgaaaaat acatcgtcac ctgggacatg
     3361 ttgcagatcc atgcacgtaa actcgcaagc cgactgatgc cttctgaaca atggaaaggc
     3421 attattgccg taagccgtgg cggtctgtac cgggtgcgtt actggcgcgt gaactgggta
     3481 ttcgtcatgt cgataccgtt tgtatttcca gctacgatca cgacaaccag cgcgagctta
     3541 aagtgctgaa acgcgcagaa ggcgatggcg aaggcttcat cgttattgat gacctggtgg
     3601 ataccggtgg tactgcggtt gcgattcgtg aaatgtatcc aaaagcgcac tttgtcacca
     3661 tcttcgcaaa accggctggt cgtccgctgg ttgatgacta tgttgttgat atcccgcaag
     3721 atacctggat tgaacagccg tgggatatgg gcgtcgtatt cgtcccgcca atctccggtc
     3781 gctaatcttt tcaacgcctg gcactgccgg gcgttgttct ttttaacttc aggcgggtta
     3841 caatagtttc cagtaagtat tctggaggct gcatccatga cacaggcaaa cctgagcgaa
     3901 accctgttca aaccccgctt taaacatcct gaaacctcga cgctagtccg ccgctttaat
     3961 cacggcgcac aaccgcctgt gcagtcggcc cttgatggta aaaccatccc tcactggtat
     4021 cgcatgatta accgtctgat gtggatctgg cgcggcattg acccacgcga aatcctcgac
     4081 gtccaggcac gtattgtgat gagcgatgcc gaacgtaccg acgatgattt atacgatacg
     4141 gtgattggct accgtggcgg caactggatt tatgagtggg ccccggatct ttgtgaagga
     4201 accttacttc tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa
     4261 ggtaaatata aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt
     4321 attttagatt ccaacctatg gaactgatga atgggagcag tggtggaatg cctttaatga
     4381 ggaaaacctg ttttgctcag aagaaatgcc atctagtgat gatgaggcta ctgctgactc
     4441 tcaacattct actcctccaa aaaagaagag aaaggtagaa gaccccaagg actttccttc
     4501 agaattgcta agttttttga gtcatgctgt gtttagtaat agaactcttg cttgctttgc
     4561 tatttacacc acaaaggaaa aagctgcact gctatacaag aaaattatgg aaaaatattc
     4621 tgtaaccttt ataagtaggc ataacagtta taatcataac atactgtttt ttcttactcc
     4681 acacaggcat agagtgtctg ctattaataa ctatgctcaa aaattgtgta cctttagctt
     4741 tttaatttgt aaaggggtta ataaggaata tttgatgtat agtgccttga ctagagatca
     4801 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     4861 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     4921 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     4981 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcaact
     5041 ggataactca agctaaccaa aatcatccca aacttcccac cccataccct attaccactg
     5101 ccaattacct gtggtttcat ttactctaaa cctgtgattc ctctgaatta ttttcatttt
     5161 aaagaaattg tatttgttaa atatgtacta caaacttagt agttggaagg gctaattcac
     5221 tcccaaagaa gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct
     5281 gattagcaga actacacacc agggccaggg gtcagatatc cactgacctt tggatggtgc
     5341 tacaagctag taccagttga gccagataag gtagaagagg ccaataaagg agagaacacc
     5401 agcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga agtgttagag
     5461 tggaggtttg acagccgcct agcatttcat cacgtggccc gagagctgca tccggagtac
     5521 ttcaagaact gctgatatcg agcttgctac aagggacttt ccgctgggga ctttccaggg
     5581 aggcgtggcc tgggcgggac tggggagtgg cgagccctca gatcctgcat ataagcagct
     5641 gctttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     5701 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     5761 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     5821 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
     5881 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
     5941 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     6001 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     6061 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     6121 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     6181 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     6241 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     6301 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cggccgctga
     6361 tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta tataaatata
     6421 aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga agagtggtgc
     6481 agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg ggagcagcag
     6541 gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa ttattgtctg
     6601 gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag catctgttgc
     6661 aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg gaaagatacc
     6721 taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt tgcaccactg
     6781 ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg aatcacacga
     6841 cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac tccttaattg
     6901 aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta gataaatggg
     6961 caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa ttattcataa
     7021 tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct atagtgaata
     7081 gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc ccgaggggac
     7141 ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac agatccattc
     7201 gattagtgaa cggatctcga cggtatcgcc aaatggcagt attcatccac aattttaaaa
     7261 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
     7321 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
     7381 acagggacag cagagatcca gtttggatcg ataagcttga tatcgaattc gggaggtggt
     7441 ccctgcagtt acgccaatga taacccccgc cagaaaaatc ttagtagcct tccctttttg
     7501 ttttccgtgc cccaactcgg cggattgact cggccccttc cggaaacacc cgaatcaact
     7561 tctagtcaaa ttattgttca cgccgcaatg acccacccct ggcccgcgtc tgtggaactg
     7621 acccctggtg tacaggagag ttcgctgctg aaagtggtcc caaaggggta ctagttttta
     7681 agctcccaac tccccctccc ccagcgtctg gaggattcca caccctcgca ccgcaggggc
     7741 gaggaagtgg gcggagtccg gttttggcgc cagccgctga ggctgccaag cagaaaagcc
     7801 accgctgagg agactccggt cactgtcctc gccccgcctc ccccttccct ccccttgggg
     7861 accaccgggc gccacgccgc gaacggtaag tgccgcggtc gtcggcgcct ccgccctccc
     7921 cctagggccc caattcccag cgggcgcggc gcgcggcccc gcgcgcagct cccggctccc
     7981 tcccccttcg gatgtggctt gagctgtagg cgcggagtcc tgcagccccg ataaaataaa
     8041 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     8101 agctagctgc agtaacgcca ttttgcaagg catggaaaaa taccaaacca agaatagaga
     8161 agttcagatc aagggcgggt acatgaaaat agctaacgtt gggccaaaca ggatatctgc
     8221 ggtgagcagt ttcggccccg gcccggggcc aagaacagat ggtcaccgca gtttcggccc
     8281 cggcccgagg ccaagaacag atggtcccca gatatggccc aaccctcagc agtttcttaa
     8341 gacccatcag atgtttccag gctcccccaa ggacctgaaa tgaccctgcg ccttatttga
     8401 attaaccaat cagcctgctt ctcgcttctg ttcgcgcgct tctgcttccc gagctctata
     8461 aaagagctca caacccctca ctcggcgcgc cagtcctccg acagactgag tcgcccgggg
     8521 gggatctgga gctctcgaga attctcacgc gtctgcagga tatcaagctt gcggtaccgc
     8581 gggcccggcc accatgaaca attcccaggg aagagtgacc ttcgaggatg tcactgtgaa
     8641 cttcacccag ggggagtggc agcggctgaa tcccgaacag agaaacttgt acagggatgt
     8701 gatgctggag aattacagca accttgtctc tgtgggacaa ggggaaacca ccaaacccga
     8761 tgtgatcttg aggttggaac aaggaaagga gccatggttg gaggaagagg aagtgctggg
     8821 aagtggccgt gcagaaaaaa atggggacat tggagggcag atttggaagc caaaggatgt
     8881 gaaagagagt ctctacccaa ctttcttgta caaagtggtt ggtggatctg gaggttctcc
     8941 caagaagaag cgcaaggtgg gacgcgtctg caggatatca agcttgcggt accgcgggcc
     9001 cgggatcgcc accatggaca agaagtacag catcggcctg gccatcggca ccaactctgt
     9061 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
     9121 caacaccgac cggcacagca tcaagaagaa cctgatcggc gccctgctgt tcgacagcgg
     9181 agaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
     9241 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
     9301 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
     9361 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
     9421 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
     9481 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
     9541 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
     9601 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
     9661 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
     9721 cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
     9781 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
     9841 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
     9901 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
     9961 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
    10021 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
    10081 gaacggctac gccggctaca tcgatggcgg agccagccag gaagagttct acaagttcat
    10141 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
    10201 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
    10261 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
    10321 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
    10381 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
    10441 gaacttcgag gaagtggtgg acaagggcgc cagcgcccag agcttcatcg agcggatgac
    10501 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
    10561 gtacttcacc gtgtacaacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
    10621 gcccgccttc ctgagcggcg agcagaaaaa agccatcgtg gacctgctgt tcaagaccaa
    10681 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
    10741 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
    10801 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
    10861 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
    10921 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
    10981 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
    11041 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
    11101 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
    11161 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
    11221 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
    11281 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
    11341 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
    11401 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
    11461 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
    11521 cgatgtggac gctatcgtgc ctcagagctt tctgaaggac gactccatcg ataacaaagt
    11581 gctgactcgg agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
    11641 gaagaagatg aagaactact ggcgccagct gctgaatgcc aagctgatta cccagaggaa
    11701 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
    11761 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
    11821 ctcccggatg aacactaagt acgacgagaa cgacaaactg atccgggaag tgaaagtgat
    11881 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
    11941 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
    12001 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
    12061 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
    12121 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
    12181 gatccggaag cggcctctga tcgagacaaa cggcgaaaca ggcgagatcg tgtgggataa
    12241 gggccgggac tttgccaccg tgcggaaagt gctgtctatg ccccaagtga atatcgtgaa
    12301 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
    12361 cgacaagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
    12421 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
    12481 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
    12541 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
    12601 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
    12661 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
    12721 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
    12781 gctgtttgtg gaacagcaca aacactacct ggacgagatc atcgagcaga tcagcgagtt
    12841 ctccaagaga gtgatcctgg ccgacgctaa tctggacaag gtgctgagcg cctacaacaa
    12901 gcacagagac aagcctatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
    12961 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
    13021 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
    13081 cgagacacgg atcgacctgt ctcagctggg aggcgacgcc tatccctatg acgtgcccga
    13141 ttatgccagc ctgggcagcg gctcccccaa gaaaaaacgc aaggtggaag atcctaagaa
    13201 aaagcggaaa gtgagcggcc gcgctactaa cttcagcctg ctgaagcagg ctggagacgt
    13261 ggaggagaac cctggacctg tgtctaaggg cgaagagctg attaaggaga acatgcacat
    13321 gaagctgtac atggagggca ccgtggacaa ccatcacttc aagtgcacat ccgagggcga
    13381 aggcaagccc tacgagggca cccagaccat gagaatcaag gtggtcgagg gcggccctct
    13441 ccccttcgcc ttcgacatcc tggctactag cttcctctac ggcagcaaga ccttcatcaa
    13501 ccacacccag ggcatccccg acttcttcaa gcagtccttc cctgagggct tcacatggga
    13561 gagagtcacc acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca
    13621 ggacggctgc ctcatctaca acgtcaagat cagaggggtg aacttcacat ccaacggccc
    13681 tgtgatgcag aagaaaacac tcggctggga ggccttcacc gagacgctgt accccgctga
    13741 cggcggcctg gaaggcagaa acgacatggc cctgaagctc gtgggcggga gccatctgat
    13801 cgcaaacgcc aagaccacat atagatccaa gaaacccgct aagaacctca agatgcctgg
    13861 cgtctactat gtggactaca gactggaaag aatcaaggag gccaacaacg agacctacgt
    13921 cgagcagcac gaggtggcag tggccagata ctgcgacctc cctagcaaac tggggcacaa
    13981 gcttaattaa gcgatcgccc tgcaggcatg caagcttgat atcaagctta tcgataatca
    14041 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
    14101 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
    14161 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
    14221 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
    14281 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
    14341 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
    14401 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
    14461 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
    14521 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
    14581 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
    14641 tcgagggaat taattcgagc tcggtacctt taagaccaat gacttacaag gcagctgtag
    14701 atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa
    14761 gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg
    14821 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
    14881 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
    14941 tttagtcagt gtggaaaatc tctagcagc
//
