LOCUS       sequence-374875-       14817 bp    DNA     circular SYN 14-APR-2025
DEFINITION  dCas9 with a C-term HA-2xNLS-XTEN80(linker)-KRAB(Kox1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14817)
  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 14817)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-EFS-dCas9-XTEN80-KRAB(Kox1)-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..14817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(64..82)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(65..82)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(372..391)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(605..624)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     724..746
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(784..802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        870..974
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(975..1043,1044..1835)
                     /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(1193..1212)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2006..2594
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2495..2514
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2748..2765
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2835..2855)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2840..3169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3020..3155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4303..4368
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4498..4518
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4943..5077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4980..4999)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5034..5053
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5245..5878
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5925..6050
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6547..6780
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6965..7009
                     /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    7294..7411
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8112..8323
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      8393..8402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8399..8425,8426..8428,8429..10915,10916..10918,
                     10919..12502)
                     /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             8399..12502
                     /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             12506..12532
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             12512..12538
                     /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             12551..12571
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12578..12598
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12908..13093
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             13103..13159
                     /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             join(13160..13162,13163..13876)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(13463..13482)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     13810..13831
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    13925..14513
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(13978..13998)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(14396..14407)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(14516..14532)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14517..14533)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(14623..14817,1..39)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catctagaat taattccgtg
       61 tattctatag tgtcacctaa atcgtatgtg tatgatacat aaggttatgt attaattgta
      121 gccgcgttct aacgacaata tgtacaagcc taattgtgta gcatctggct tactgaagca
      181 gaccctatca tctctctcgt aaactgccgt cagagtcggt ttggttggac gaaccttctg
      241 agtttctggt aacgccgtcc cgcacccgga aatggtcagc gaaccaatca gcagggtcat
      301 cgctagccag atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
      361 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
      421 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
      481 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
      541 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgaa tggtgcactc
      601 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      661 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
      721 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
      781 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      841 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      901 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      961 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1021 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1081 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1141 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1201 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1261 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1321 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1381 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1441 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1501 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1561 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1621 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1681 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1741 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1801 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1861 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1921 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1981 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2041 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2101 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2161 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2221 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2281 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2341 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2401 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2461 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2521 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2581 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2641 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2701 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2761 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2821 attaatgcag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2941 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3001 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3061 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3121 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttggaca
     3181 caagacaggc ttgcgagata tgtttgagaa taccacttta tcccgcgtca gggagaggca
     3241 gtgcgtaaaa agacgcggac tcatgtgaaa tactggtttt tagtgcgcca gatctctata
     3301 atctcgcgca acctattttc ccctcgaaca ctttttaagc cgtagataaa caggctggga
     3361 cacttcacat gagcgaaaaa tacatcgtca cctgggacat gttgcagatc catgcacgta
     3421 aactcgcaag ccgactgatg ccttctgaac aatggaaagg cattattgcc gtaagccgtg
     3481 gcggtctgta ccgggtgcgt tactggcgcg tgaactgggt attcgtcatg tcgataccgt
     3541 ttgtatttcc agctacgatc acgacaacca gcgcgagctt aaagtgctga aacgcgcaga
     3601 aggcgatggc gaaggcttca tcgttattga tgacctggtg gataccggtg gtactgcggt
     3661 tgcgattcgt gaaatgtatc caaaagcgca ctttgtcacc atcttcgcaa aaccggctgg
     3721 tcgtccgctg gttgatgact atgttgttga tatcccgcaa gatacctgga ttgaacagcc
     3781 gtgggatatg ggcgtcgtat tcgtcccgcc aatctccggt cgctaatctt ttcaacgcct
     3841 ggcactgccg ggcgttgttc tttttaactt caggcgggtt acaatagttt ccagtaagta
     3901 ttctggaggc tgcatccatg acacaggcaa acctgagcga aaccctgttc aaaccccgct
     3961 ttaaacatcc tgaaacctcg acgctagtcc gccgctttaa tcacggcgca caaccgcctg
     4021 tgcagtcggc ccttgatggt aaaaccatcc ctcactggta tcgcatgatt aaccgtctga
     4081 tgtggatctg gcgcggcatt gacccacgcg aaatcctcga cgtccaggca cgtattgtga
     4141 tgagcgatgc cgaacgtacc gacgatgatt tatacgatac ggtgattggc taccgtggcg
     4201 gcaactggat ttatgagtgg gccccggatc tttgtgaagg aaccttactt ctgtggtgtg
     4261 acataattgg acaaactacc tacagagatt taaagctcta aggtaaatat aaaattttta
     4321 agtgtataat gtgttaaact actgattcta attgtttgtg tattttagat tccaacctat
     4381 ggaactgatg aatgggagca gtggtggaat gcctttaatg aggaaaacct gttttgctca
     4441 gaagaaatgc catctagtga tgatgaggct actgctgact ctcaacattc tactcctcca
     4501 aaaaagaaga gaaaggtaga agaccccaag gactttcctt cagaattgct aagttttttg
     4561 agtcatgctg tgtttagtaa tagaactctt gcttgctttg ctatttacac cacaaaggaa
     4621 aaagctgcac tgctatacaa gaaaattatg gaaaaatatt ctgtaacctt tataagtagg
     4681 cataacagtt ataatcataa catactgttt tttcttactc cacacaggca tagagtgtct
     4741 gctattaata actatgctca aaaattgtgt acctttagct ttttaatttg taaaggggtt
     4801 aataaggaat atttgatgta tagtgccttg actagagatc ataatcagcc ataccacatt
     4861 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa
     4921 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag
     4981 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5041 gtccaaactc atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca
     5101 aaatcatccc aaacttccca ccccataccc tattaccact gccaattacc tgtggtttca
     5161 tttactctaa acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta
     5221 aatatgtact acaaacttag tagttggaag ggctaattca ctcccaaaga agacaagata
     5281 tccttgatct gtggatctac cacacacaag gctacttccc tgattagcag aactacacac
     5341 cagggccagg ggtcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg
     5401 agccagataa ggtagaagag gccaataaag gagagaacac cagcttgtta caccctgtga
     5461 gcctgcatgg gatggatgac ccggagagag aagtgttaga gtggaggttt gacagccgcc
     5521 tagcatttca tcacgtggcc cgagagctgc atccggagta cttcaagaac tgctgatatc
     5581 gagcttgcta caagggactt tccgctgggg actttccagg gaggcgtggc ctgggcggga
     5641 ctggggagtg gcgagccctc agatcctgca tataagcagc tgctttttgc ctgtactggg
     5701 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5761 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5821 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     5881 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
     5941 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
     6001 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
     6061 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     6121 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     6181 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     6241 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     6301 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     6361 acaaaagtaa gaccaccgca cagcaagcgg ccggccgctg atcttcagac ctggaggagg
     6421 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     6481 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     6541 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     6601 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     6661 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     6721 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     6781 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     6841 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     6901 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     6961 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     7021 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     7081 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     7141 accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat
     7201 agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg
     7261 acggtatcgc caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     7321 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     7381 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     7441 agtttggatc gataagcttg atatcgaatt cgggaggtgg tccctgcagt tacgccaatg
     7501 ataacccccg ccagaaaaat cttagtagcc ttcccttttt gttttccgtg ccccaactcg
     7561 gcggattgac tcggcccctt ccggaaacac ccgaatcaac ttctagtcaa attattgttc
     7621 acgccgcaat gacccacccc tggcccgcgt ctgtggaact gacccctggt gtacaggaga
     7681 gttcgctgct gaaagtggtc ccaaaggggt actagttttt aagctcccaa ctccccctcc
     7741 cccagcgtct ggaggattcc acaccctcgc accgcagggg cgaggaagtg ggcggagtcc
     7801 ggttttggcg ccagccgctg aggctgccaa gcagaaaagc caccgctgag gagactccgg
     7861 tcactgtcct cgccccgcct cccccttccc tccccttggg gaccaccggg cgccacgccg
     7921 ckaacggtaa gtgccgcggt cgtcggcgcc tccgccctcc ccctagggcc ccaattccca
     7981 gcgggcgcgg cgcgcggccc cgcgcgcagc tcccggctcc ctcccccttc ggatgtggct
     8041 tgagctgtag gcgcggagtc ctgcagccta ggtcttgaaa ggagtgggaa ttggctccgg
     8101 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     8161 cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     8221 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     8281 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggggggat ctggagctct
     8341 cgagaattct cacgcgtctg caggatatca agcttgcggt accgcgggcc cggccaccat
     8401 ggacaagaag tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat
     8461 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
     8521 cagcatcaag aagaacctga tcggcgccct gctgttcgac agcggagaaa cagccgaggc
     8581 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     8641 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     8701 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     8761 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     8821 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     8881 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     8941 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     9001 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     9061 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggcaacct
     9121 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     9181 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     9241 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     9301 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     9361 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     9421 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     9481 ctacatcgat ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     9541 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     9601 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     9661 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     9721 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     9781 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     9841 ggtggacaag ggcgccagcg cccagagctt catcgagcgg atgaccaact tcgataagaa
     9901 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     9961 caacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
    10021 cggcgagcag aaaaaagcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
    10081 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
    10141 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
    10201 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
    10261 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
    10321 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag
    10381 gctgagccgg aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga
    10441 tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag
    10501 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
    10561 cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt
    10621 gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
    10681 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat
    10741 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
    10801 ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga
    10861 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggacgctat
    10921 cgtgcctcag agctttctga aggacgactc catcgataac aaagtgctga ctcggagcga
    10981 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
    11041 ctactggcgc cagctgctga atgccaagct gattacccag aggaagttcg acaatctgac
    11101 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
    11161 ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
    11221 taagtacgac gagaacgaca aactgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
    11281 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
    11341 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
    11401 ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
    11461 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
    11521 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc
    11581 tctgatcgag acaaacggcg aaacaggcga gatcgtgtgg gataagggcc gggactttgc
    11641 caccgtgcgg aaagtgctgt ctatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
    11701 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgaca agctgatcgc
    11761 cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta
    11821 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa
    11881 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
    11941 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
    12001 ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca
    12061 gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca
    12121 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
    12181 gcacaaacac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
    12241 cctggccgac gctaatctgg acaaggtgct gagcgcctac aacaagcaca gagacaagcc
    12301 tatcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
    12361 tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga
    12421 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga
    12481 cctgtctcag ctgggaggcg acgcctatcc ctatgacgtg cccgattatg ccagcctggg
    12541 cagcggctcc cccaagaaaa aacgcaaggt ggaagatcct aagaaaaagc ggaaagtgga
    12601 cggcattggt agtgggagca acggcagcag cggatccgga gggccgagct ctggcgcacc
    12661 cccaccaagt ggagggtctc ctgccgggtc cccaacatct actgaagaag gcaccagcga
    12721 atccgcaacg cccgagtcag gccctggtac ctccacagaa ccatctgaag gtagtgcgcc
    12781 tggttcccca gctggaagcc ctacttccac cgaagaaggc acgtcaaccg aaccaagtga
    12841 aggatctgcc cctgggacca gcactgaacc atctgagatg gatgctaagt cactaactgc
    12901 ctggtcccgg acactggtga ccttcaagga tgtatttgtg gacttcacca gggaggagtg
    12961 gaagctgctg gacactgctc agcagatcgt gtacagaaat gtgatgctgg agaactataa
    13021 gaacctggtt tccttgggtt atcagcttac taagccagat gtgatcctcc ggttggagaa
    13081 gggagaagag cccagcggcc gcgctactaa cttcagcctg ctgaagcagg ctggagacgt
    13141 ggaggagaac cctggacctg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
    13201 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtctg gcgagggcga
    13261 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
    13321 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
    13381 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
    13441 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
    13501 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
    13561 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
    13621 cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg
    13681 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
    13741 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
    13801 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
    13861 cgagctgtac aagtaataag cgatcgccct gcaggcatgc aagcttgata tcaagcttat
    13921 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
    13981 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
    14041 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
    14101 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
    14161 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
    14221 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
    14281 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
    14341 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
    14401 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
    14461 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
    14521 ccgtcgacct cgagggaatt aattcgagct cggtaccttt aagaccaatg acttacaagg
    14581 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
    14641 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
    14701 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
    14761 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagat
//
