LOCUS       sequence-271550-       10864 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid encoding the Cas9 gene and a gRNA expression cassette 
            allowing to clone a single or multiple gRNAs for DSBs induction in 
            yeast. Constructed by Aubin Fleiss.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10864)
  AUTHORS   Agier N, Fleiss A, Delmas S, Fischer G
  TITLE     A Versatile Protocol to Generate Translocations in Yeast Genomes 
            Using CRISPR/Cas9.
  JOURNAL   Methods Mol Biol. 2021;2196:181-198. doi: 
            10.1007/978-1-0716-0868-5_14.
  PUBMED    32889721
REFERENCE   2  (bases 1 to 10864)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2021"; volume: "2196:181-198. doi"; pages: " 
            10.1007/978-1-0716-0868-5_14"
COMMENT     Sequence Label: pAEF5
FEATURES             Location/Qualifiers
     source          1..10864
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    93..114
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(129..134,135..152,153..159)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    167..183
                     /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)."
     primer_bind     172..194
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     191..207
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     191..207
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     226..246
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        228..246
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        264..662
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     primer_bind     666..682
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     666..682
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             694..4797
                     /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             694..4797
                     /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             694..4797
                     /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             4810..4830
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4846..5093
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4862..4880)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        5115..5383
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5404..5478
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5483..5502
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(5524..5543)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5525..5543)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5553..5570)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5553..5569)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5562..5584)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     gene            5674..7249
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        5674..6017
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6018..7046
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             6018..7046
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      7052..7249
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     7457..7479
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7517..7535)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      7576..8918
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7908..7955
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        8945..9049
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9050..9118,9119..9910)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(9268..9287)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10081..10669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10570..10589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10823..10840
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
       61 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
      121 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      181 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
      241 taaagggaac aaaagctgga gctcatagct tcaaaatgtt tctactcctt ttttactctt
      301 ccagattttc tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata
      361 ctaaatttcc cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga
      421 aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt
      481 atcacgtttc tttttcttga aaattttttt tttgattttt ttctctttcg atgacctccc
      541 attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc atttttcttg
      601 ttctattaca acttttttta cttcttgctc attagaaaga aagcatagca atctaatcta
      661 agttttctag aactagtgga tcccccggga aaaatggaca agaagtactc cattgggctc
      721 gatatcggca caaacagcgt cggttgggcc gtcattacgg acgagtacaa ggtgccgagc
      781 aaaaaattca aagttctggg caataccgat cgccacagca taaagaagaa cctcattggc
      841 gccctcctgt tcgactccgg ggagacggcc gaagccacgc ggctcaaaag aacagcacgg
      901 cgcagatata cccgcagaaa gaatcggatc tgctacctgc aggagatctt tagtaatgag
      961 atggctaagg tggatgactc tttcttccat aggctggagg agtccttttt ggtggaggag
     1021 gataaaaagc acgagcgcca cccaatcttt ggcaatatcg tggacgaggt ggcgtaccat
     1081 gaaaagtacc caaccatata tcatctgagg aagaagcttg tagacagtac tgataaggct
     1141 gacttgcggt tgatctatct cgcgctggcg catatgatca aatttcgggg acacttcctc
     1201 atcgaggggg acctgaaccc agacaacagc gatgtcgaca aactctttat ccaactggtt
     1261 cagacttaca atcagctttt cgaagagaac ccgatcaacg catccggagt tgacgccaaa
     1321 gcaatcctga gcgctaggct gtccaaatcc cggcggctcg aaaacctcat cgcacagctc
     1381 cctggggaga agaagaacgg cctgtttggt aatcttatcg ccctgtcact cgggctgacc
     1441 cccaacttta aatctaactt cgacctggcc gaagatgcca agcttcaact gagcaaagac
     1501 acctacgatg atgatctcga caatctgctg gcccagatcg gcgaccagta cgcagacctt
     1561 tttttggcgg caaagaacct gtcagacgcc attctgctga gtgatattct gcgagtgaac
     1621 acggagatca ccaaagctcc gctgagcgct agtatgatca agcgctatga tgagcaccac
     1681 caagacttga ctttgctgaa ggcccttgtc agacagcaac tgcctgagaa gtacaaggaa
     1741 attttcttcg atcagtctaa aaatggctac gccggataca ttgacggcgg agcaagccag
     1801 gaggaatttt acaaatttat taagcccatc ttggaaaaaa tggacggcac cgaggagctg
     1861 ctggtaaagc ttaacagaga agatctgttg cgcaaacagc gcactttcga caatggaagc
     1921 atcccccacc agattcacct gggcgaactg cacgctatcc tcaggcggca agaggatttc
     1981 tacccctttt tgaaagataa cagggaaaag attgagaaaa tcctcacatt tcggataccc
     2041 tactatgtag gccccctcgc ccggggaaat tccagattcg cgtggatgac tcgcaaatca
     2101 gaagagacca tcactccctg gaacttcgag gaagtcgtgg ataagggggc ctctgcccag
     2161 tccttcatcg aaaggatgac taactttgat aaaaatctgc ctaacgaaaa ggtgcttcct
     2221 aaacactctc tgctgtacga gtacttcaca gtttataacg agctcaccaa ggtcaaatac
     2281 gtcacagaag ggatgagaaa gccagcattc ctgtctggag agcagaagaa agctatcgtg
     2341 gacctcctct tcaagacgaa ccggaaagtt accgtgaaac agctcaaaga agactatttc
     2401 aaaaagattg aatgtttcga ctctgttgaa atcagcggag tggaggatcg cttcaacgca
     2461 tccctgggaa cgtatcacga tctcctgaaa atcattaaag acaaggactt cctggacaat
     2521 gaggagaacg aggacattct tgaggacatt gtcctcaccc ttacgttgtt tgaagatagg
     2581 gagatgattg aagaacgctt gaaaacttac gctcatctct tcgacgacaa agtcatgaaa
     2641 cagctcaaga ggcgccgata tacaggatgg gggcggctgt caagaaaact gatcaatggg
     2701 atccgagaca agcagagtgg aaagacaatc ctggattttc ttaagtccga tggatttgcc
     2761 aaccggaact tcatgcagtt gatccatgat gactctctca cctttaagga ggacatccag
     2821 aaagcacaag tttctggcca gggggacagt cttcacgagc acatcgctaa tcttgcaggt
     2881 agcccagcta tcaaaaaggg aatactgcag accgttaagg tcgtggatga actcgtcaaa
     2941 gtaatgggaa ggcataagcc cgagaatatc gttatcgaga tggcccgaga gaaccaaact
     3001 acccagaagg gacagaagaa cagtagggaa aggatgaaga ggattgaaga gggtataaaa
     3061 gaactggggt cccaaatcct taaggaacac ccagttgaaa acacccagct tcagaatgag
     3121 aagctctacc tgtactacct gcagaacggc agggacatgt acgtggatca ggaactggac
     3181 atcaatcggc tctccgacta cgacgtggat catatcgtgc cccagtcttt tctcaaagat
     3241 gattctattg ataataaagt gttgacaaga tccgataaaa atagagggaa gagtgataac
     3301 gtcccctcag aagaagttgt caagaaaatg aaaaattatt ggcggcagct gctgaacgcc
     3361 aaactgatca cacaacggaa gttcgataat ctgactaagg ctgaacgagg tggcctgtct
     3421 gagttggata aagccggctt catcaaaagg cagcttgttg agacacgcca gatcaccaag
     3481 cacgtggccc aaattctcga ttcacgcatg aacaccaagt acgatgaaaa tgacaaactg
     3541 attcgagagg tgaaagttat tactctgaag tctaagctgg tctcagattt cagaaaggac
     3601 tttcagtttt ataaggtgag agagatcaac aattaccacc atgcgcatga tgcctacctg
     3661 aatgcagtgg taggcactgc acttatcaaa aaatatccca agcttgaatc tgaatttgtt
     3721 tacggagact ataaagtgta cgatgttagg aaaatgatcg caaagtctga gcaggaaata
     3781 ggcaaggcca ccgctaagta cttcttttac agcaatatta tgaatttttt caagaccgag
     3841 attacactgg ccaatggaga gattcggaag cgaccactta tcgaaacaaa cggagaaaca
     3901 ggagaaatcg tgtgggacaa gggtagggat ttcgcgacag tccggaaggt cctgtccatg
     3961 ccgcaggtga acatcgttaa aaagaccgaa gtacagaccg gaggcttctc caaggaaagt
     4021 atcctcccga aaaggaacag cgacaagctg atcgcacgca aaaaagattg ggaccccaag
     4081 aaatacggcg gattcgattc tcctacagtc gcttacagtg tactggttgt ggccaaagtg
     4141 gagaaaggga agtctaaaaa actcaaaagc gtcaaggaac tgctgggcat cacaatcatg
     4201 gagcgatcaa gcttcgaaaa aaaccccatc gactttctcg aggcgaaagg atataaagag
     4261 gtcaaaaaag acctcatcat taagcttccc aagtactctc tctttgagct tgaaaacggc
     4321 cggaaacgaa tgctcgctag tgcgggcgag ctgcagaaag gtaacgagct ggcactgccc
     4381 tctaaatacg ttaatttctt gtatctggcc agccactatg aaaagctcaa agggtctccc
     4441 gaagataatg agcagaagca gctgttcgtg gaacaacaca aacactacct tgatgagatc
     4501 atcgagcaaa taagcgaatt ctccaaaaga gtgatcctcg ccgacgctaa cctcgataag
     4561 gtgctttctg cttacaataa gcacagggat aagcccatca gggagcaggc agaaaacatt
     4621 atccacttgt ttactctgac caacttgggc gcgcctgcag ccttcaagta cttcgacacc
     4681 accatagaca gaaagcggta cacctctaca aaggaggtcc tggacgccac actgattcat
     4741 cagtcaatta cggggctcta tgaaacaaga atcgacctct ctcagctcgg tggagacagc
     4801 agggctgacc ccaagaagaa gaggaaggtg tgatctcttc tcgagtcatg taattagtta
     4861 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     4921 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     4981 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     5041 tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcggccggt
     5101 accgcgggcc cgcttctttg aaaagataat gtatgattat gctttcactc atatttatac
     5161 agaaacttga tgttttcttt cgagtatata caaggtgatt acatgtacgt ttgaagtaca
     5221 actctagatt ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt tttcacaccc
     5281 tacaatgttc tgttcaaaag attttggtca aacgctgtag aagtgaaagt tggtgcgcat
     5341 gtttcggcgt tcgaaacttc tccgcagtga aagataaatg atcagaagag ccatggctct
     5401 tcagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     5461 agtggcaccg agtcggtggt gctttttttg ttttttatgt ctgcggccgc ggtacccaat
     5521 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     5581 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatcccag atctgtttag
     5641 cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt
     5701 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
     5761 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
     5821 aaaaattacg gctcctcgct gcggacctgc gagcagggaa acgctcccct cacagacgcg
     5881 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
     5941 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
     6001 tccgaacata aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     6061 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     6121 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     6181 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     6241 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     6301 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     6361 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
     6421 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     6481 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     6541 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     6601 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     6661 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     6721 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     6781 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     6841 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     6901 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     6961 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     7021 gcactcgtcc gagggcaaag gaataatcag tactgacaat aaaaagattc ttgttttcaa
     7081 gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag
     7141 cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca
     7201 gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg
     7261 atactaacgc cgccatccag tgtcgaaaac gagctcgaat tcatcgatga tatcagatcc
     7321 actagtggcc tatgcggccc aatctgctct gatgccgcat agttaagcca gccccgacac
     7381 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     7441 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     7501 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     7561 atggtttctt agtatgatcc aatatcaaag gaaatgatag cattgaagga tgagactaat
     7621 ccaattgagg agtggcagca tatagaacag ctaaagggta gtgctgaagg aagcatacga
     7681 taccccgcat ggaatgggat aatatcacag gaggtactag actacctttc atcctacata
     7741 aatagacgca tataagtacg catttaagca taaacacgca ctatgccgtt cttctcatgt
     7801 atatatatat acaggcaaca cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc
     7861 gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa acgctttgaa gttcctattc
     7921 cgaagttcct attctctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg
     7981 ctctgaagac gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat
     8041 attgcgaata ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt
     8101 gctatatccc tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc
     8161 gacctctaca ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta
     8221 agaactattc atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag
     8281 tatatagaga caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa
     8341 cgctatcact ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg
     8401 gatgccttta tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa
     8461 gtggagtcag gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc
     8521 ttaacggacc tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag
     8581 aaaaaaagta atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta
     8641 gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg
     8701 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
     8761 ttgttttaca aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat
     8821 ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg
     8881 catttttgtt ctacaaaatg aagcacagat gcttcgttca ggtggcactt ttcggggaaa
     8941 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     9001 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     9061 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     9121 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     9181 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     9241 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     9301 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     9361 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     9421 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     9481 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     9541 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     9601 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     9661 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     9721 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     9781 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     9841 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     9901 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     9961 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
    10021 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
    10081 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
    10141 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
    10201 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
    10261 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
    10321 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
    10381 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
    10441 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
    10501 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
    10561 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
    10621 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
    10681 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
    10741 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
    10801 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaga gacgcccaat
    10861 acgc
//
