LOCUS       sequence-308961-       13306 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses Cas9 at very low level.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13306)
  AUTHORS   Port F, Strein C, Stricker M, Rauscher B, Heigwer F, Zhou J, 
            Beyersdorffer C, Frei J, Hess A, Kern K, Lange L, Langner N, Malamud
            R, Pavlovic B, Radecke K, Schmitt L, Voos L, Valentini E, Boutros M
  TITLE     A large-scale resource for tissue-specific CRISPR mutagenesis in 
            Drosophila.
  JOURNAL   Elife. 2020 Feb 13;9. pii: 53865. doi: 10.7554/eLife.53865.
  PUBMED    32053108
REFERENCE   2  (bases 1 to 13306)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2020-02-13"; volume: "9. pii: 53865. doi"; pages: " 
            10.7554/eLife.53865"
COMMENT     Sequence Label: UAS-u(XXL)Cas9
FEATURES             Location/Qualifiers
     source          1..13306
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(675..693)
                     /label=pCasper-F
                     /note="Drosophila mini-white gene, reverse primer"
     CDS             join(1145..1216,1618..1891,1966..2620,2682..2997,3201..3332,
                     3403..4017)
                     /codon_start=1
                     /gene="white"
                     /product="Drosophila white gene eye color pigment"
                     /label=mini-white
                     /note="This is a modified version of the white gene lacking
                     part of the first intron."
                     /translation="MGQEDQELLIRGGSKHPSAEHLNNGDSGAASQSCINQGFGQAKNY
                     GTLRPPSPPEDSGSGSGQLAENLTYAWHNMDIFGAVNQPGSGWRQLVNRTRGLFCNERH
                     IPAPRKHLLKNVCGVAYPGELLAVMGSSGAGKTTLLNALAFRSPQGIQVSPSGMRLLNG
                     QPVDAKEMQARCAYVQQDDLFIGSLTAREHLIFQAMVRMPRHLTYRQRVARVDQVIQEL
                     SLSKCQHTIIGVPGRVKGLSGGERKRLAFASEALTDPPLLICDEPTSGLDSFTAHSVVQ
                     VLKKLSQKGKTVILTIHQPSSELFELFDKILLMAEGRVAFLGTPSEAVDFFSYVGAQCP
                     TNYNPADFYVQVLAVVPGREIESRDRIAKICDNFAISKVARDMEQLLATKNLEKPLEQP
                     ENGYTYKATWFMQFRAVLWRSWLSVLKEPLLVKVRLIQTTMVAILIGLIFLGQQLTQVG
                     VMNINGAIFLFLTNMTFQNVFATINVFTSELPVFMREARSRLYRCDTYFLGKTIAELPL
                     FLTVPLVFTAIAYPMIGLRAGVLHFFNCLALVTLVANVSTSFGYLISCASSSTSMALSV
                     GPPVIIPFLLFGGFFLNSGSVPVYLKWLSYLSWFRYANEGLLINQWADVEPGEISCTSS
                     NTTCPSSGKVILETLNFSAADLPLDYVGLAILIVSFRVLAYLALRLRARRKE"
     protein_bind    4792..4825
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4858..4952
                     /label=5X UAS
                     /bound_moiety="GAL4"
                     /note="five tandem copies of the ""ScaI site"" 17-mer 
                     CGGAGTACTGTCCTCCG, an upstream activating sequence (UAS) 
                     that efficiently binds yeast Gal4 (Webster et al., 1988; 
                     Pfeiffer et al., 2010)"
     promoter        4971..5209
                     /label=hsp70 promoter
                     /note="Drosophila melanogaster hsp70Bb promoter"
     primer_bind     5156..5177
                     /label=pCasper-hs
                     /note="Drosophila Hsp70 promoter, forward primer"
     CDS             join(5233..5235,5236..5238,5239..5952)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSMQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5278..5299)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5539..5558)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5886..5907
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5957..10060
                     /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             5957..10060
                     /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             5957..10060
                     /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             10073..10093
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          10180..10245
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             10375..10395
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    10667..10801
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10704..10723)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10758..10777
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    10830..10899
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     promoter        complement(11118..11136)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(11157..11173)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11157..11173)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11170..11192)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11181..11197
                     /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(11205..11211,11212..11229,11230..11235))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11250..11271
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11388..11405)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11559..12147)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11639..11658)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12318..13109,13110..13178))
                     /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     12941..12960
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13179..13283)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtacgtaccg
      661 ggcccctagt atgtatgtaa gttaataaaa cccatttttg cggaaagtag ataaaaaaaa
      721 catttttttt ttttactgca ctggatatca ttgaacttat ctgatcagtt ttaaatttac
      781 ttcgatccaa gggtatttga tgtaccaggt tctttcgatt acctctcact caaaatgaca
      841 ttccactcaa agtcagcgct gtttgcctcc ttctctgtcc acagaaatat cgccgtctct
      901 ttcgccgctg cgtccgctat ctctttcgcc accgtttgta gcgttacgta gcgtcaatgt
      961 ccgccttcag ttgcattttg tcagcggttt cgtgacgaag ctccaagcgg tttacgccat
     1021 caattaaaca caaagtgctg tgccaaaact cctctcgctt cttatttttg tttgtttttt
     1081 gagtgattgg ggtggtgatt ggttttgggt gggtaagcag gggaaagtgt gaaaaatccc
     1141 ggcaatgggc caagaggatc aggagctatt aattcgcgga ggcagcaaac acccatctgc
     1201 cgagcatctg aacaatgtga gtagtacatg tgcatacatc ttaagttcac ttgatctata
     1261 ggaactgcga ttgcaacatc aaattgtctg cggcgtgaga actgcgaccc acaaaaatcc
     1321 caaaccgcaa ttgcacaaac aaatagtgac acgaaacaga ttattctggt agctgttctc
     1381 gctatataag acaatttttg agatcatatc atgatcaaga catctaaagg cattcatttt
     1441 cgactatatt cttttttaca aaaaatataa caaccagata ttttaagctg atcctagatg
     1501 cacaaaaaat aaataaaagt ataaacctac ttcgtaggat acttcggggt actttttgtt
     1561 cggggttaga tgagcataac gcttgtagtt gatatttgag atcccctatc attgcagggt
     1621 gacagcggag cggcttcgca gagctgcatt aaccagggct tcgggcaggc caaaaactac
     1681 ggcacgctcc ggccacccag tccgccggag gactccggtt cagggagcgg ccaactagcc
     1741 gagaacctca cctatgcctg gcacaatatg gacatctttg gggcggtcaa tcagccgggc
     1801 tccggatggc ggcagctggt caaccggaca cgcggactat tctgcaacga gcgacacata
     1861 ccggcgccca ggaaacattt gctcaagaac ggtgagtttc tattcgcagt cggctgatct
     1921 gtgtgaaatc ttaataaagg gtccaattac caatttgaaa ctcagtttgc ggcgtggcct
     1981 atccgggcga acttttggcc gtgatgggca gttccggtgc cggaaagacg accctgctga
     2041 atgcccttgc ctttcgatcg ccgcagggca tccaagtatc gccatccggg atgcgactgc
     2101 tcaatggcca acctgtggac gccaaggaga tgcaggccag gtgcgcctat gtccagcagg
     2161 atgacctctt tatcggctcc ctaacggcca gggaacacct gattttccaa gccatggtgc
     2221 ggatgccacg acatctgacc tatcggcagc gagtggcccg cgtggatcag gtgatccagg
     2281 agctttcgct cagcaaatgt cagcacacga tcatcggtgt gcccggcagg gtgaaaggtc
     2341 tgtccggcgg agaaaggaag cgtctggcat tcgcctccga ggctctaacc gatccgccgc
     2401 ttctgatctg cgatgagccc acctccggac tggactcctt taccgcccac agcgtcgtcc
     2461 aggtgctgaa gaagctgtcg cagaagggca agaccgtcat cctgaccatt catcagccgt
     2521 cttccgagct gtttgagctc tttgacaaga tccttctgat ggccgagggc agggtagctt
     2581 tcttgggcac tcccagcgaa gccgtcgact tcttttccta gtgagttcga tgtgtttatt
     2641 aagggtatct agtattacat aacatctcaa ctcctatcca gcgtgggtgc ccagtgtcct
     2701 accaactaca atccggcgga cttttacgta caggtgttgg ccgttgtgcc cggacgggag
     2761 atcgagtccc gtgatcggat cgccaagata tgcgacaatt ttgccattag caaagtagcc
     2821 cgggatatgg agcagttgtt ggccaccaaa aatctggaga agccactgga gcagccggag
     2881 aatgggtaca cctacaaggc cacctggttc atgcagttcc gggcggtcct gtggcgatcc
     2941 tggctgtcgg tgctcaagga accactcctc gtaaaagtgc gacttattca gacaacggtg
     3001 agtggttcca gtggaaacaa atgatataac gcttacaatt cttggaaaca aattcgctag
     3061 attttagata gaattgcctg attccacacc cttcttagtt tttttcaatg agatgtatag
     3121 tttatagttt tgcagaagat aaataaattt catttaactc gcgaatatta atgagatgcg
     3181 agtaacattt taatttgcag atggttgcca tcttgattgg cctcatcttt ttgggccaac
     3241 aactcacgca agtgggtgtg atgaatatca acggagccat cttcctcttc ctgaccaaca
     3301 tgacctttca aaacgtcttt gccacgataa atgtaagtca tgtttagaat acatttgcat
     3361 ttcaataatt tactaacttt ctaatgaatc gattcgattt aggtgttcac ctcagagctg
     3421 ccagttttta tgagggaggc ccgaagtcga ctttatcgct gtgacacata ctttctgggc
     3481 aaaacgattg ccgaattgcc gctttttctc acagtgccac tggtcttcac ggcgattgcc
     3541 tatccgatga tcggactgcg ggccggagtg ctgcacttct tcaactgcct ggcgctggtc
     3601 actctggtgg ccaatgtgtc aacgtccttc ggatatctaa tatcctgcgc cagctcctcg
     3661 acctcgatgg cgctgtctgt gggtccgccg gttatcatac cattcctgct ctttggcggc
     3721 ttcttcttga actcgggctc ggtgccagta tacctcaaat ggttgtcgta cctctcatgg
     3781 ttccgttacg ccaacgaggg tctgctgatt aaccaatggg cggacgtgga gccgggcgaa
     3841 attagctgca catcgtcgaa caccacgtgc cccagttcgg gcaaggtcat cctggagacg
     3901 cttaacttct ccgccgccga tctgccgctg gactacgtgg gtctggccat tctcatcgtg
     3961 agcttccggg tgctcgcata tctggctcta agacttcggg cccgacgcaa ggagtagccg
     4021 acatatatcc gaaataactg cttgtttttt ttttttacca ttattaccat cgtgtttact
     4081 gtttattgcc ccctcaaaaa gctaatgtaa ttatatttgt gccaataaaa acaagatatg
     4141 acctatagaa tacaagtatt tccccttcga acatccccac aagtagactt tggatttgtc
     4201 ttctaaccaa aagacttaca cacctgcata ccttacatca aaaactcgtt tatcgctaca
     4261 taaaacaccg ggatatattt tttatataca tacttttcaa atcgcgcgcc ctcttcataa
     4321 ttcacctcca ccacaccacg tttcgtagtt gctctttcgc tgtctcccac ccgctctccg
     4381 caacacattc accttttgtt cgacgacctt ggagcgactg tcgttagttc cgcgcgattc
     4441 ggttcgctca aatggttccg agtggttcat ttcgtctcaa tagaaattag taataaatat
     4501 ttgtatgtac aatttatttg ctccaatata tttgtatata tttccctcac agctatattt
     4561 attctaattt aatattatga ctttttaagg taattttttg tgacctgttc ggagtgatta
     4621 gcgttacaat ttgaactgaa agtgacatcc agtgtttgtt ccttgtgtag atgcatctca
     4681 aaaaaatggt gggcataata gtgttgttta tatatatcaa aaataacaac tataataata
     4741 agaatacatt taatttagaa aatgcttgga tttcactgga actaggctag cataacttcg
     4801 tataatgtat gctatacgaa gttatgctag cggatccaag cttgcatgcc tgcaggtcgg
     4861 agtactgtcc tccgagcgga gtactgtcct ccgagcggag tactgtcctc cgagcggagt
     4921 actgtcctcc gagcggagta ctgtcctccg agcggagact ctagcgagcg ccggagtata
     4981 aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa acaagcaaag tgaacacgtc
     5041 gctaagcgaa agctaagcaa ataaacaagc gcagctgaac aagctaaaca atctgcagta
     5101 aagtgcaagt taaagtgaat caattaaaag taaccagcaa ccaagtaaat caactgcaac
     5161 tactgaaatc tgccaagaag taattattga atacaagaag agaactctga atagggaatt
     5221 gggaattcca acatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     5281 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     5341 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     5401 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     5461 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     5521 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     5581 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     5641 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     5701 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     5761 atgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     5821 cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc
     5881 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     5941 ctgtacaagt aataacatgg acaagaagta ctccattggg ctcgatatcg gcacaaacag
     6001 cgtcggctgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct
     6061 gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc
     6121 cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag
     6181 aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga
     6241 ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg
     6301 ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat
     6361 atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta
     6421 tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa
     6481 cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct
     6541 tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag
     6601 gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa
     6661 cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa
     6721 cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct
     6781 cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa
     6841 cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc
     6901 tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct
     6961 gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc
     7021 taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt
     7081 tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag
     7141 agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca
     7201 cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga
     7261 taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg taggccccct
     7321 cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc
     7381 ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat
     7441 gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta
     7501 cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag
     7561 aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac
     7621 gaaccggaaa gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt
     7681 cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca
     7741 cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat
     7801 tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg
     7861 cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg
     7921 atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag
     7981 tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca
     8041 gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg
     8101 ccagggggac agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa
     8161 gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa
     8221 gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa
     8281 gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat
     8341 ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta
     8401 cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga
     8461 ctacgacgtg gatcatatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa
     8521 agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct cagaagaagt
     8581 tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg
     8641 gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg
     8701 cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct
     8761 cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt
     8821 tattactctg aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt
     8881 gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac
     8941 tgcacttatc aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt
     9001 gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa
     9061 gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg
     9121 agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga
     9181 caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt
     9241 taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa
     9301 cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga
     9361 ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa
     9421 aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga
     9481 aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat
     9541 cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc
     9601 tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt
     9661 cttgtatctg gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa
     9721 gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga
     9781 attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa
     9841 taagcacagg gataagccca tcagggagca ggcagaaaac attatccact tgtttactct
     9901 gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg
     9961 gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct
    10021 ctatgaaaca agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa
    10081 gaagaggaag gtgtgagcta gcggatcttt gtgaaggaac cttacttctg tggtgtgaca
    10141 taattggaca aactacctac agagatttaa agctctaagg taaatataaa atttttaagt
    10201 gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc aacctatgga
    10261 actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt ttgctcagaa
    10321 gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac tcctccaaaa
    10381 aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag ttttttgagt
    10441 catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac aaaggaaaaa
    10501 gctgcactgc tatacaagaa aattatggaa aaatatttga tgtatagtgc cttgactaga
    10561 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
    10621 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
    10681 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
    10741 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
    10801 ccactagtgt cgacgatgta ggtcacggtc tcgaagccgc ggtgcgggtg ccagggcgtg
    10861 cccttgggct ccccgggcgc gtactccacc tcacccatct ggtccatcat gatgaacggg
    10921 tcgaggtggc ggtagttgat cccggcgaac gcgcggcgca ccgggaagcc ctcgccctcg
    10981 aaaccgctgg gcgcggtggt cacggtgagc acgggacgtg cgacggcgtc ggcgggtgcg
    11041 gatacgcggg gcagcgtcag cgggttctcg acggtcacgg cgggcatgtc gacactagtt
    11101 ctagccagct tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
    11161 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
    11221 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
    11281 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
    11341 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
    11401 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
    11461 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
    11521 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
    11581 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
    11641 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
    11701 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
    11761 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
    11821 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
    11881 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
    11941 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
    12001 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
    12061 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
    12121 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
    12181 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
    12241 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
    12301 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
    12361 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
    12421 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
    12481 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
    12541 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
    12601 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
    12661 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
    12721 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
    12781 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
    12841 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
    12901 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
    12961 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
    13021 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
    13081 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
    13141 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
    13201 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
    13261 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccac
//
