LOCUS       sequence-374877-       15613 bp    DNA     circular SYN 15-DEC-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 15613)
  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 15613)
  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-dCas9-XTEN80-KRAB(Kox1)-IRES-mCh...
FEATURES             Location/Qualifiers
     source          1..15613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(77..95)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(78..95)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(385..404)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(618..637)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     737..759
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(797..815)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        883..987
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(988..1056,1057..1848)
                     /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(1206..1225)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2019..2607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2508..2527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2761..2778
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2848..2868)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2853..3182
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3033..3168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          4316..4381
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4511..4531
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4956..5090
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4993..5012)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5047..5066
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             5258..5891
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5938..6063
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6560..6793
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6978..7022
                     /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    7307..7424
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        8166..8573
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      8642..8651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(8648..8674,8675..8677,8678..11164,11165..11167,
                     11168..12751)
                     /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             8648..12751
                     /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             12755..12781
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             12761..12787
                     /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             12800..12820
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             12827..12847
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             13157..13342
                     /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"
     misc_feature    13381..13948
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    13398..13950
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(13542..13559)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     13769..13788
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             13966..14676
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(14111..14129)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     14379..14398
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    14734..15322
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(14787..14807)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(15205..15216)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(15325..15341)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(15326..15342)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             join(15432..15613,1..52)
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
ORIGIN
        1 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagcatctag
       61 aattaattcc gtgtattcta tagtgtcacc taaatcgtat gtgtatgata cataaggtta
      121 tgtattaatt gtagccgcgt tctaacgaca atatgtacaa gcctaattgt gtagcatctg
      181 gcttactgaa gcagacccta tcatctctct cgtaaactgc cgtcagagtc ggtttggttg
      241 gacgaacctt ctgagtttct ggtaacgccg tcccgcaccc ggaaatggtc agcgaaccaa
      301 tcagcagggt catcgctagc cagatcctct acgccggacg catcgtggcc ggcatcaccg
      361 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
      421 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
      481 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
      541 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
      601 gaatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
      661 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
      721 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
      781 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
      841 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
      901 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
      961 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1021 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1081 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1141 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1201 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1261 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1321 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     1381 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     1441 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     1501 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     1561 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     1621 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     1681 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     1741 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     1801 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     1861 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1921 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1981 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2041 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2101 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2161 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2221 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2281 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2341 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2401 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2461 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2521 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2581 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2641 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2701 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2761 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     2821 cgttggccga ttcattaatg cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2881 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2941 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3001 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3061 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3121 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3181 aaaagcttgg acacaagaca ggcttgcgag atatgtttga gaataccact ttatcccgcg
     3241 tcagggagag gcagtgcgta aaaagacgcg gactcatgtg aaatactggt ttttagtgcg
     3301 ccagatctct ataatctcgc gcaacctatt ttcccctcga acacttttta agccgtagat
     3361 aaacaggctg ggacacttca catgagcgaa aaatacatcg tcacctggga catgttgcag
     3421 atccatgcac gtaaactcgc aagccgactg atgccttctg aacaatggaa aggcattatt
     3481 gccgtaagcc gtggcggtct gtaccgggtg cgttactggc gcgtgaactg ggtattcgtc
     3541 atgtcgatac cgtttgtatt tccagctacg atcacgacaa ccagcgcgag cttaaagtgc
     3601 tgaaacgcgc agaaggcgat ggcgaaggct tcatcgttat tgatgacctg gtggataccg
     3661 gtggtactgc ggttgcgatt cgtgaaatgt atccaaaagc gcactttgtc accatcttcg
     3721 caaaaccggc tggtcgtccg ctggttgatg actatgttgt tgatatcccg caagatacct
     3781 ggattgaaca gccgtgggat atgggcgtcg tattcgtccc gccaatctcc ggtcgctaat
     3841 cttttcaacg cctggcactg ccgggcgttg ttctttttaa cttcaggcgg gttacaatag
     3901 tttccagtaa gtattctgga ggctgcatcc atgacacagg caaacctgag cgaaaccctg
     3961 ttcaaacccc gctttaaaca tcctgaaacc tcgacgctag tccgccgctt taatcacggc
     4021 gcacaaccgc ctgtgcagtc ggcccttgat ggtaaaacca tccctcactg gtatcgcatg
     4081 attaaccgtc tgatgtggat ctggcgcggc attgacccac gcgaaatcct cgacgtccag
     4141 gcacgtattg tgatgagcga tgccgaacgt accgacgatg atttatacga tacggtgatt
     4201 ggctaccgtg gcggcaactg gatttatgag tgggccccgg atctttgtga aggaacctta
     4261 cttctgtggt gtgacataat tggacaaact acctacagag atttaaagct ctaaggtaaa
     4321 tataaaattt ttaagtgtat aatgtgttaa actactgatt ctaattgttt gtgtatttta
     4381 gattccaacc tatggaactg atgaatggga gcagtggtgg aatgccttta atgaggaaaa
     4441 cctgttttgc tcagaagaaa tgccatctag tgatgatgag gctactgctg actctcaaca
     4501 ttctactcct ccaaaaaaga agagaaaggt agaagacccc aaggactttc cttcagaatt
     4561 gctaagtttt ttgagtcatg ctgtgtttag taatagaact cttgcttgct ttgctattta
     4621 caccacaaag gaaaaagctg cactgctata caagaaaatt atggaaaaat attctgtaac
     4681 ctttataagt aggcataaca gttataatca taacatactg ttttttctta ctccacacag
     4741 gcatagagtg tctgctatta ataactatgc tcaaaaattg tgtaccttta gctttttaat
     4801 ttgtaaaggg gttaataagg aatatttgat gtatagtgcc ttgactagag atcataatca
     4861 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     4921 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     4981 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5041 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc aactggataa
     5101 ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc actgccaatt
     5161 acctgtggtt tcatttactc taaacctgtg attcctctga attattttca ttttaaagaa
     5221 attgtatttg ttaaatatgt actacaaact tagtagttgg aagggctaat tcactcccaa
     5281 agaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattag
     5341 cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg gtgctacaag
     5401 ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa caccagcttg
     5461 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt agagtggagg
     5521 tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga gtacttcaag
     5581 aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc agggaggcgt
     5641 ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc agctgctttt
     5701 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     5761 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     5821 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     5881 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
     5941 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
     6001 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
     6061 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
     6121 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
     6181 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     6241 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     6301 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     6361 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca
     6421 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     6481 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     6541 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     6601 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     6661 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     6721 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     6781 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     6841 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     6901 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     6961 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     7021 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     7081 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     7141 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     7201 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     7261 tgaacggatc tcgacggtat cgccaaatgg cagtattcat ccacaatttt aaaagaaaag
     7321 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
     7381 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
     7441 acagcagaga tccagtttgg atcgataagc ttgatatcga attcgggagg tggtccctgc
     7501 agttacgcca atgataaccc ccgccagaaa aatcttagta gccttccctt tttgttttcc
     7561 gtgccccaac tcggcggatt gactcggccc cttccggaaa cacccgaatc aacttctagt
     7621 caaattattg ttcacgccgc aatgacccac ccctggcccg cgtctgtgga actgacccct
     7681 ggtgtacagg agagttcgct gctgaaagtg gtcccaaagg ggtactagtt tttaagctcc
     7741 caactccccc tcccccagcg tctggaggat tccacaccct cgcaccgcag gggcgaggaa
     7801 gtgggcggag tccggttttg gcgccagccg ctgaggctgc caagcagaaa agccaccgct
     7861 gaggagactc cggtcactgt cctcgccccg cctccccctt ccctcccctt ggggaccacc
     7921 gggcgccacg ccgcgaacgg taagtgccgc ggtcgtcggc gcctccgccc tccccctagg
     7981 gccccaattc ccagcgggcg cggcgcgcgg ccccgcgcgc agctcccggc tccctccccc
     8041 ttcggatgtg gcttgagctg taggcgcgga gtcctgcagc cccgataaaa taaaagattt
     8101 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag
     8161 ctgcagtaac gccattttgc aaggcatgga aaaataccaa accaagaata gagaagttca
     8221 gatcaagggc gggtacatga aaatagctaa cgttgggcca aacaggatat ctgcggtgag
     8281 cagtttcggc cccggcccgg ggccaagaac agatggtcac cgcagtttcg gccccggccc
     8341 gaggccaaga acagatggtc cccagatatg gcccaaccct cagcagtttc ttaagaccca
     8401 tcagatgttt ccaggctccc ccaaggacct gaaatgaccc tgcgccttat ttgaattaac
     8461 caatcagcct gcttctcgct tctgttcgcg cgcttctgct tcccgagctc tataaaagag
     8521 ctcacaaccc ctcactcggc gcgccagtcc tccgacagac tgagtcgccc gggggggatc
     8581 tggagctctc gagaattctc acgcgtctgc aggatatcaa gcttgcggta ccgcgggccc
     8641 ggccaccatg gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg
     8701 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     8761 cgaccggcac agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggagaaac
     8821 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     8881 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     8941 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     9001 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     9061 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     9121 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     9181 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     9241 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     9301 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     9361 cggcaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     9421 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     9481 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     9541 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     9601 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     9661 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     9721 ctacgccggc tacatcgatg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     9781 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     9841 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     9901 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     9961 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
    10021 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
    10081 cgaggaagtg gtggacaagg gcgccagcgc ccagagcttc atcgagcgga tgaccaactt
    10141 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
    10201 caccgtgtac aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
    10261 cttcctgagc ggcgagcaga aaaaagccat cgtggacctg ctgttcaaga ccaaccggaa
    10321 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
    10381 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
    10441 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
    10501 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
    10561 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
    10621 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
    10681 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
    10741 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
    10801 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
    10861 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
    10921 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
    10981 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
    11041 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
    11101 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
    11161 ggacgctatc gtgcctcaga gctttctgaa ggacgactcc atcgataaca aagtgctgac
    11221 tcggagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
    11281 gatgaagaac tactggcgcc agctgctgaa tgccaagctg attacccaga ggaagttcga
    11341 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
    11401 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
    11461 gatgaacact aagtacgacg agaacgacaa actgatccgg gaagtgaaag tgatcaccct
    11521 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
    11581 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
    11641 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
    11701 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
    11761 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
    11821 gaagcggcct ctgatcgaga caaacggcga aacaggcgag atcgtgtggg ataagggccg
    11881 ggactttgcc accgtgcgga aagtgctgtc tatgccccaa gtgaatatcg tgaaaaagac
    11941 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgacaa
    12001 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
    12061 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
    12121 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
    12181 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
    12241 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
    12301 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
    12361 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
    12421 tgtggaacag cacaaacact acctggacga gatcatcgag cagatcagcg agttctccaa
    12481 gagagtgatc ctggccgacg ctaatctgga caaggtgctg agcgcctaca acaagcacag
    12541 agacaagcct atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
    12601 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
    12661 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
    12721 acggatcgac ctgtctcagc tgggaggcga cgcctatccc tatgacgtgc ccgattatgc
    12781 cagcctgggc agcggctccc ccaagaaaaa acgcaaggtg gaagatccta agaaaaagcg
    12841 gaaagtggac ggcattggta gtgggagcaa cggcagcagc ggatccggag ggccgagctc
    12901 tggcgcaccc ccaccaagtg gagggtctcc tgccgggtcc ccaacatcta ctgaagaagg
    12961 caccagcgaa tccgcaacgc ccgagtcagg ccctggtacc tccacagaac catctgaagg
    13021 tagtgcgcct ggttccccag ctggaagccc tacttccacc gaagaaggca cgtcaaccga
    13081 accaagtgaa ggatctgccc ctgggaccag cactgaacca tctgagatgg atgctaagtc
    13141 actaactgcc tggtcccgga cactggtgac cttcaaggat gtatttgtgg acttcaccag
    13201 ggaggagtgg aagctgctgg acactgctca gcagatcgtg tacagaaatg tgatgctgga
    13261 gaactataag aacctggttt ccttgggtta tcagcttact aagccagatg tgatcctccg
    13321 gttggagaag ggagaagagc cctagtaagc ggccgcttag atctctcgag gttaacgaat
    13381 tccgcccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
    13441 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
    13501 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
    13561 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
    13621 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
    13681 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
    13741 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
    13801 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
    13861 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
    13921 ggttttcctt tgaaaaacac gatgataata tggccacaac catggatggt gagcaagggc
    13981 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
    14041 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
    14101 acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc
    14161 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
    14221 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
    14281 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
    14341 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc
    14401 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
    14461 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
    14521 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
    14581 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
    14641 cactccaccg gcggcatgga cgagctgtac aagtaaggcc gcgactctag agtcgacctg
    14701 caggcatgca agcttgatat caagcttatc gataatcaac ctctggatta caaaatttgt
    14761 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
    14821 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
    14881 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
    14941 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
    15001 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
    15061 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
    15121 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
    15181 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
    15241 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
    15301 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gagggaatta attcgagctc
    15361 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
    15421 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
    15481 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
    15541 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
    15601 gttgtgtgac tct
//
