LOCUS       sequence-245962-       13472 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of a guide RNA targeting an intergenic 
            region of human chromosome 2 (CRISPR cutting control) and S. 
            pyogenes Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13472)
  AUTHORS   Chan EM, Shibue T, McFarland JM, Gaeta B, Ghandi M, Dumont N, 
            Gonzalez A, McPartlan JS, Li T, Zhang Y, Bin Liu J, Lazaro JB, Gu P,
            Piett CG, Apffel A, Ali SO, Deasy R, Keskula P, Ng RWS, Roberts EA, 
            Reznichenko E, Leung L, Alimova M, Schenone M, Islam M, Maruvka YE, 
            Liu Y, Roper J, Raghavan S, Giannakis M, Tseng YY, Nagel ZD, 
            D'Andrea A, Root DE, Boehm JS, Getz G, Chang S, Golub TR, Tsherniak 
            A, Vazquez F, Bass AJ
  TITLE     WRN helicase is a synthetic lethal target in microsatellite unstable
            cancers.
  JOURNAL   Nature. 2019 Apr 10. pii: 10.1038/s41586-019-1102-x. doi: 
            10.1038/s41586-019-1102-x.
  PUBMED    30971823
REFERENCE   2  (bases 1 to 13472)
  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: "Nature. 
            2019 Apr 10. pii: 10.1038/s41586-019-1102-x. doi: 
            10.1038/s41586-019-1102-x."
COMMENT     Sequence Label: pXPR_BRD051-sgCh2-2
FEATURES             Location/Qualifiers
     source          1..13472
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    47..172
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    665..898
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1083..1127
                     /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    1425..1542
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1593..1841
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1593..1613
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1764..1783
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2010..2221
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2240..2249
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2246..6349
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             2246..6349
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6350..6397
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6398..6421
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6431..6487
                     /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             6497..7522
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     misc_feature    7544..8132
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7597..7617)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8015..8026)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8210..8443
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8468..8485)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8474..8698
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8744..9172
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8831..8850)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9041..9062
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9181..9201)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9186..9515
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9366..9501
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9428..9447
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9563..9610
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9629..10003
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    10133..10254
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10170..10189)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10224..10243
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10303..10319)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10303..10319)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10316..10338)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10327..10343
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(10351..10357,10358..10375,10376..10381))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10396..10417
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10534..10551)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10705..11293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10785..10804)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11464..12255,12256..12324))
                     /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     12087..12106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12325..12429)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12504..12523)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        12695..13074
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        13076..13274
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     13229..13249
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             13292..13472
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag
       61 gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc
      121 aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa
      181 gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa
      241 atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc
      301 tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct
      361 tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt
      421 gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca
      481 aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag
      541 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
      601 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
      661 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
      721 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
      781 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
      841 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
      901 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
      961 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     1021 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     1081 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     1141 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     1201 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     1261 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     1321 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     1381 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     1441 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     1501 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     1561 agcagagatc cagtttggtt aattaaggta ccgagggcct atttcccatg attccttcat
     1621 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     1681 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1741 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1801 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgggtgtgcg tatgaagcag
     1861 tggtttcaga gctacagcag aaatgctgta gcaagttgaa ataaggctag tccgttatca
     1921 acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc gctagctagg tcttgaaagg
     1981 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
     2041 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
     2101 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
     2161 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
     2221 ggaccggttc tagagcgctg ccaccatgga caagaagtac agcatcggcc tggacatcgg
     2281 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     2341 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     2401 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     2461 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     2521 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     2581 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     2641 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     2701 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     2761 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     2821 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     2881 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     2941 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     3001 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     3061 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     3121 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     3181 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     3241 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     3301 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     3361 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     3421 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     3481 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     3541 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     3601 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     3661 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     3721 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     3781 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     3841 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     3901 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     3961 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     4021 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     4081 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     4141 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     4201 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     4261 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     4321 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     4381 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     4441 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     4501 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     4561 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     4621 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     4681 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     4741 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     4801 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     4861 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     4921 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     4981 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     5041 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     5101 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     5161 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     5221 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     5281 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     5341 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     5401 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     5461 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     5521 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     5581 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     5641 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     5701 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     5761 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     5821 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     5881 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     5941 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     6001 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     6061 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     6121 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     6181 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     6241 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     6301 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc
     6361 cgccacaaag aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa
     6421 gggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc
     6481 tggaccgggc ggatcgatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     6541 gatcgaaaag ttcgacagcg tgtccgacct gatgcagctc tcggagggcg aagaatctcg
     6601 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     6661 tggtttctac aaagatcgtt atgttcatcg gcactttgca tcggccgcgc tcccgattcc
     6721 ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct cccgccgtgc
     6781 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     6841 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     6901 attcggaccg caaggaatcg gtcaatacac tacgtggcgt gatttcatat gcgcgattgc
     6961 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     7021 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     7081 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     7141 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     7201 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     7261 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     7321 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     7381 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     7441 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     7501 tcgtccgagg gcaaaggaat gagttaacac gcgttaagtc gacaatcaac ctctggatta
     7561 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     7621 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     7681 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     7741 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     7801 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     7861 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     7921 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     7981 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     8041 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     8101 gagtcggatc tccctttggg ccgcctcccc gcctcgaggt cgactttaag accaatgact
     8161 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     8221 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     8281 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     8341 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     8401 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggccgt ttaaacccgc
     8461 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     8521 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     8581 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     8641 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     8701 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     8761 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     8821 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     8881 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     8941 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     9001 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     9061 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     9121 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     9181 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     9241 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     9301 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     9361 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     9421 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     9481 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     9541 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt
     9601 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct
     9661 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg
     9721 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag
     9781 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct
     9841 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg
     9901 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc
     9961 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt
    10021 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
    10081 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
    10141 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
    10201 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
    10261 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
    10321 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
    10381 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
    10441 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
    10501 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
    10561 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
    10621 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
    10681 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
    10741 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
    10801 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
    10861 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
    10921 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
    10981 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
    11041 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
    11101 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
    11161 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
    11221 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
    11281 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
    11341 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
    11401 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
    11461 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
    11521 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
    11581 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
    11641 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
    11701 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
    11761 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
    11821 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
    11881 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
    11941 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
    12001 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
    12061 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
    12121 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
    12181 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
    12241 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
    12301 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
    12361 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
    12421 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
    12481 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
    12541 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
    12601 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
    12661 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
    12721 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
    12781 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
    12841 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
    12901 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
    12961 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
    13021 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
    13081 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
    13141 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
    13201 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
    13261 gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc agatctgagc
    13321 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
    13381 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
    13441 acccttttag tcagtgtgga aaatctctag ca
//
