LOCUS       sequence-240808-       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"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3025..3236
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3255..3264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3261..7364
                     /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             3261..7364
                     /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             7365..7412
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7413..7436
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7446..7502
                     /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             7512..8537
                     /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    8559..9147
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8612..8632)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9030..9041)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             9225..9458
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9483..9500)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9489..9713
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9759..10187
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9846..9865)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10056..10077
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10196..10216)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10201..10530
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10381..10516
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10443..10462
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10578..10625
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10644..11018
                     /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    11148..11269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11185..11204)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11239..11258
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11318..11334)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11318..11334)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11331..11353)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11342..11358
                     /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(11366..11372,11373..11390,11391..11396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11411..11432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11549..11566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11720..12308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11800..11819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12479..13270,13271..13339))
                     /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     13102..13121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13340..13444)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccgggt gtgcgtatga agcagtggtt
     2881 tcagagctac agcagaaatg ctgtagcaag ttgaaataag gctagtccgt tatcaacttg
     2941 aaaaagtggc accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg
     3001 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     3061 tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg
     3121 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     3181 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc
     3241 ggttctagag cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca
     3301 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     3361 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     3421 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     3481 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     3541 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     3601 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     3661 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     3721 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     3781 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     3841 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     3901 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     3961 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     4021 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     4081 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     4141 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     4201 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     4261 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     4321 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     4381 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     4441 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     4501 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     4561 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     4621 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     4681 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     4741 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     4801 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     4861 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     4921 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     4981 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     5041 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     5101 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     5161 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     5221 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     5281 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     5341 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     5401 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     5461 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     5521 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     5581 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     5641 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     5701 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     5761 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     5821 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     5881 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     5941 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     6001 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     6061 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     6121 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     6181 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     6241 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     6301 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     6361 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     6421 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     6481 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     6541 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     6601 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     6661 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     6721 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     6781 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     6841 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     6901 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     6961 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     7021 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     7081 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     7141 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     7201 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     7261 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     7321 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca
     7381 caaagaaggc tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat
     7441 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     7501 cgggcggatc gatgaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     7561 aaaagttcga cagcgtgtcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     7621 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     7681 tctacaaaga tcgttatgtt catcggcact ttgcatcggc cgcgctcccg attccggaag
     7741 tgcttgacat tggggaattt agcgagagcc tgacctattg catctcccgc cgtgcacagg
     7801 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     7861 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     7921 gaccgcaagg aatcggtcaa tacactacgt ggcgtgattt catatgcgcg attgctgatc
     7981 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     8041 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     8101 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     8161 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     8221 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     8281 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     8341 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     8401 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     8461 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     8521 cgagggcaaa ggaatgagtt aacacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     8581 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     8641 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     8701 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     8761 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     8821 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     8881 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     8941 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     9001 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     9061 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     9121 ggatctccct ttgggccgcc tccccgcctc gaggtcgact ttaagaccaa tgacttacaa
     9181 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     9241 ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     9301 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     9361 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     9421 cctcagaccc ttttagtcag tgtggaaaat ctctagcagg gccgtttaaa cccgctgatc
     9481 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     9541 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     9601 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     9661 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     9721 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     9781 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     9841 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     9901 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     9961 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
    10021 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
    10081 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
    10141 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
    10201 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
    10261 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
    10321 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
    10381 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
    10441 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
    10501 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
    10561 ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat
    10621 acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg gtgctcaccg
    10681 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact
    10741 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg
    10801 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg
    10861 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg
    10921 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg
    10981 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga gatttcgatt
    11041 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
    11101 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
    11161 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
    11221 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
    11281 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
    11341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
    11401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
    11461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
    11521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
    11581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
    11641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
    11701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
    11761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
    11821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
    11881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
    11941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
    12001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
    12061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
    12121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
    12181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
    12241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
    12301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
    12361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
    12421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
    12481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
    12541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
    12601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
    12661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
    12721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
    12781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
    12841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
    12901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
    12961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
    13021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
    13081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
    13141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
    13201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
    13261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
    13321 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
    13381 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
    13441 cgcgcacatt tccccgaaaa gtgccacctg ac
//
