LOCUS       sequence-270597-       13783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses drosophila-optimized Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13783)
  AUTHORS   Viswanatha R, Li Z, Hu Y, Perrimon N
  TITLE     Pooled genome-wide CRISPR screening for basal and context-specific 
            fitness gene essentiality in Drosophila cells.
  JOURNAL   Elife. 2018 Jul 27;7. pii: 36333. doi: 10.7554/eLife.36333.
  PUBMED    30051818
REFERENCE   2  (bases 1 to 13783)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2018-07-27"; volume: "7. pii: 36333. doi"; pages: " 
            10.7554/eLife.36333"
COMMENT     Sequence Label: pMK33/Cas9
FEATURES             Location/Qualifiers
     source          1..13783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(624..641)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(795..1383)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(875..894)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1554..2345,2346..2414))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2177..2196
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2415..2519)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2587..2605
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4001..4280
                     /label=copia promoter
                     /note="strong promoter from the Drosophila transposable 
                     element copia (Sinclair et al., 1986)"
     CDS             4326..5351
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4326..5351
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     intron          5714..5779
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             5909..5929
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    6354..6488
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6391..6410)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6445..6464
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        6489..6915
                     /label=MT promoter
                     /note="Drosophila metallothionein promoter"
     primer_bind     6923..6939
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     6924..6940
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6955..6979
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      6994..7003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7003..7068
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7075..7095
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7120..11220
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7120..11220
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             11221..11268
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(11302..11319)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11308..11515
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(11553..11577)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             11600..12346
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             12365..12394
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             12398..12427
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             12431..12460
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
ORIGIN
        1 cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa
       61 ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat cgtcacggcg
      121 atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta
      181 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga
      241 atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat
      301 tcttgcggag aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg
      361 ccatctccag cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc
      421 gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt
      481 agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg
      541 cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc
      601 ggaagtcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
      661 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      721 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      781 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      841 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      901 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      961 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1021 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1081 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1141 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1201 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     1261 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1321 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1381 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1441 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1501 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1561 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1621 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1681 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1741 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     1801 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     1861 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     1921 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     1981 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2041 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2101 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2161 ccggcgtcaa cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2221 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2281 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2341 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2401 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2461 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2521 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     2581 tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattcacatt tgtacgaatt
     2641 ttttttttat caaaagttcg agtttttcac caatttcctc atcaaccgag caaggcaaac
     2701 ggctttgaat aatatggtgt tatatataca tatatcaaat cgctgctgac tgcgtgattg
     2761 atggccccaa gattacatat tatcgaatca ggattcagaa ggagatcaat gtcaaatgcg
     2821 gacaggaaca tgaaagacgc ctgttatgcg caattaaaaa tttgggttta attgctgtgg
     2881 aaactgttgt tggcggcatc ttaagttcct gtttaacaac atcaactact tatgtacgta
     2941 gaagcgttta agccatttgc atacagatga gaactggctt ttgtgctaat cagtcaagat
     3001 gactccgatg atgatgactc attacctgac cagttttcgc tgctttcttt tcaacaacta
     3061 cttgtatatg tattgtatcc aatagcaata cattgaattt ccatggtcta gtcacgtatt
     3121 atcatttaat tgacaccaag tcgtgttatt gttgagctat cgagttcagc tcaaacattt
     3181 cttattccca tgaataagcc ggcaaaaata tgcaatctat gaaagttaat ataagcaaac
     3241 cttactttga ctcaatacca atgcactttg tgtcgatagg ttcacgcaat tgaggcgatt
     3301 attccgataa cccaagcgat tgactgttcc cgtttcgatt ccaattgaaa tttggaaatg
     3361 tacaatagtt ttgctatatg ctgtcaagta cgctcttatc ttctctgggt tttcttcaga
     3421 gtttcgaaac gcttcttctt ttttttgttt tttttttttt ggaatctcgt attttggaag
     3481 gggctcccct ctggaatttg ttacactgtc gttatcattg cgaacaagcg gcccgaagct
     3541 atcagcgact ttaacattta caatgcactt ttttacgacc aattaaatgt acattttcct
     3601 ttcttcgccc gttgataagc gaacgcgatg tggcgcaggc aatgtgttgc tcttgcgaca
     3661 caaacgcaat caaaatggat tcaatttcgc tttttcccag tgaaacgaag aacgaaccga
     3721 ccatcatgat atgctcctct gcatgttgcg tattgaatca atgacaattt caattaagcc
     3781 gcccgttcgt catgcgtttt cgtgcgcttc gaaatgctga taacgctgct gtcctccaac
     3841 tgctttgcat gtggacacaa ttccatttat ttaattcttt tatttggatc ggttaaatta
     3901 aaaagcgcct tgttacgcat ttaacgttgt ttccggtgcg tggtggtttc atgcttctgg
     3961 gaacggcaaa tgggtttagg attgggaacc cctcatcatc tgttggaata tactattcaa
     4021 cctacaaaag taacgttaaa caacactact ttatatttga tatgaatggc cacacctttt
     4081 atgccataaa acatattgta agagaatacc actcttttta ttccttcttt ccttcttgta
     4141 cgttttttgc tgtaagtagg tcgtggtgct ggtgttgcag ttgaaataac ttaaaatata
     4201 aatcataaaa ctcaaacata aacttgacta tttatttatt tattaagaaa ggaaatataa
     4261 attataaatt acaacaggtt atggacctgc agccaagctt ggcgctcgtc cgggggcaat
     4321 gagatatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt
     4381 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct
     4441 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca
     4501 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg
     4561 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
     4621 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca
     4681 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc
     4741 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg
     4801 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg
     4861 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt
     4921 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
     4981 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt
     5041 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat
     5101 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg
     5161 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat
     5221 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg
     5281 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
     5341 caaaggaata gagtagatgc cgaccgaaca agagctgatt tcgagaacgc ctcagccagc
     5401 aactcgcgcg agcctagcaa ggcaaatgcg agagaacggc cttacgcttg gtggcacagt
     5461 tctcgtccac agttcgctaa gctcgctcgg ctgggtcgcg ggagggccgg tcgcagtgat
     5521 tcaggccctt ctggattgtg ttggtcccca gggcacgatt gtcatgccca cgcactcggg
     5581 tgatctgact gatcccgcag attggagatc gccgcccgtg cctgccgatt gggtgcagat
     5641 ctttgtgaag gaaccttact tctgtggtgt gacataattg gacaaactac ctacagagat
     5701 ttaaagctct aaggtaaata taaaattttt aagtgtataa tgtgttaaac tactgattct
     5761 aattgtttgt gtattttaga ttccaaccta tggaactgat gaatgggagc agtggtggaa
     5821 tgcctttaat gaggaaaacc tgttttgctc agaagaaatg ccatctagtg atgatgaggc
     5881 tactgctgac tctcaacatt ctactcctcc aaaaaagaag agaaaggtag aagaccccaa
     5941 ggactttcct tcagaattgc taagtttttt gagtcatgct gtgtttagta atagaactct
     6001 tgcttgcttt gctatttaca ccacaaagga aaaagctgca ctgctataca agaaaattat
     6061 ggaaaaatat tctgtaacct ttataagtag gcataacagt tataatcata acatactgtt
     6121 ttttcttact ccacacaggc atagagtgtc tgctattaat aactatgctc aaaaattgtg
     6181 tacctttagc tttttaattt gtaaaggggt taataaggaa tatttgatgt atagtgcctt
     6241 gactagagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     6301 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     6361 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     6421 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     6481 tctggatcaa ttcgttgcag gacaggatgt ggtgcccgat gtgactagct ctttgctgca
     6541 ggccgtccta tcctctggtt ccgataagag acccagaact ccggcccccc accgcccacc
     6601 gccaccccca tacatatgtg gtacgcaagt aagagtgcct gcgcatgccc catgtgcccc
     6661 accaagagct ttgcatccca tacaagtccc caaagtggag aaccgaacca attcttcgcg
     6721 ggcagaacaa aagcttctgc acacgtctcc actcgaattt ggagccggcc ggcgtgtgca
     6781 aaagaggtga atcgaacgaa agacccgtgt gtaaagccgc gtttccaaaa tgtataaaac
     6841 cgagagcatc tggccaatgt gcatcagttg tggtcagcag caaaatcaag tgaatcatct
     6901 cagtgcaact aaagggggga tctcgaggtc gacggtatcg ataagcttga tatcacaagt
     6961 ttgtacaaaa aagcaggctc cgaattcgcc cttgccacca tggattacaa ggatcacgat
     7021 ggcgactaca aggaccatga catcgactat aaggacgacg acgataagat ggcccccaag
     7081 aagaagcgca aagtgggcat ccatggcgtg ccagccgccg ataagaagta cagcatcggc
     7141 ctggatatcg gcaccaacag cgtgggctgg gccgtgatca ccgatgagta caaggtgccc
     7201 agcaagaagt tcaaggtgct gggcaacacc gatcgccaca gcatcaagaa gaacctgatc
     7261 ggcgccctgc tgttcgatag cggagaaacc gccgaggcca cccgcctgaa gcgcaccgcc
     7321 cgccgccgct atacccgccg caagaatcgc atctgctatc tgcaagagat cttcagcaac
     7381 gagatggcca aggtggacga tagcttcttc caccgcctgg aggagtcctt cctggtggag
     7441 gaggataaga agcacgagcg ccaccccatc ttcggcaaca tcgtggatga ggtggcctac
     7501 cacgagaagt accccaccat ctaccatctg cgcaagaagc tggtggatag caccgataag
     7561 gccgatctgc gcctgatcta cctggccctg gcccacatga tcaagttccg cggccacttc
     7621 ctgatcgagg gcgatctgaa ccccgataac tccgatgtgg ataagctgtt catccagctg
     7681 gtgcagacct acaaccagct gttcgaggag aaccccatca acgccagcgg cgtggacgcc
     7741 aaggccattc tgagtgcccg cctgagcaag agtcgccgcc tggagaatct gatcgcccag
     7801 ctgccaggcg agaagaagaa cggcctgttc ggcaacctga ttgccctgag cctgggcctg
     7861 acccccaact tcaagagcaa cttcgatctg gccgaggatg ccaagctgca gctgagcaag
     7921 gatacctacg atgatgatct ggataacctg ctggcccaga tcggcgatca gtacgccgac
     7981 ctgtttctgg ccgccaagaa cctgtccgat gccatcctgc tgagcgacat cctgcgcgtg
     8041 aacaccgaga tcaccaaggc cccactgagc gcctcgatga tcaagcgcta cgatgagcac
     8101 caccaggatc tgaccctgct gaaggccctc gtgcgccagc agctgcccga gaagtataag
     8161 gagatcttct tcgaccagag caagaacggc tacgccggct acatcgatgg cggagccagc
     8221 caggaggagt tctacaagtt catcaagccc atcctggaga agatggatgg caccgaggag
     8281 ctgctcgtga agctgaaccg cgaggatctg ctgcgcaagc agcgcacctt cgataacggc
     8341 agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcgccg ccaggaggat
     8401 ttttacccgt tcctgaagga taaccgcgag aagattgaga agatcctgac cttccggatc
     8461 ccctactacg tgggaccact ggcccgcggc aatagccgct ttgcctggat gacccgcaag
     8521 agcgaggaaa ccatcacccc ctggaacttc gaggaggtgg tggataaggg cgccagcgcc
     8581 cagagcttca tcgagcgcat gaccaacttc gacaagaatc tgcccaacga gaaggtgctg
     8641 cccaagcaca gcctgctgta cgagtacttc accgtgtaca acgagctgac gaaagtgaaa
     8701 tacgtgaccg agggaatgcg caagcccgcc ttcctgagcg gagagcagaa gaaggccatc
     8761 gtggacctgc tgtttaagac caaccgcaaa gtgaccgtga agcagctgaa ggaggactac
     8821 ttcaagaaga tcgagtgctt cgacagcgtg gagatctcgg gcgtggagga tcgcttcaat
     8881 gccagcctgg gcacgtacca cgatctgctg aagattatca aggacaagga cttcctggac
     8941 aacgaggaga atgaggacat tctggaggac atcgtgctga ccctgaccct gtttgaggat
     9001 cgcgagatga tcgaggagcg cctcaagacc tacgcccatc tgtttgatga caaagtgatg
     9061 aagcagctga agcgccgccg ctacaccgga tggggacgcc tgagtcgcaa gctgatcaac
     9121 ggcatccgcg ataagcagag cggcaagacg atcctggatt tcctgaagtc cgatggcttc
     9181 gccaatcgca acttcatgca gctgatccac gacgattcgc tgaccttcaa ggaggatatt
     9241 cagaaggccc aggtgtccgg ccagggcgat agcctgcatg agcacattgc caacctggcc
     9301 ggcagccccg ccattaagaa gggcattctg cagaccgtga aggtggtgga cgagctcgtg
     9361 aaagtgatgg gccgccacaa gcccgagaat atcgtgattg agatggcccg cgagaaccag
     9421 accacccaga agggccagaa gaacagccgc gagcgcatga agcgcatcga ggagggcatt
     9481 aaggagctgg gcagccagat tctgaaggag caccccgtgg agaacaccca gctgcagaac
     9541 gagaagctgt acctgtacta cctgcagaac ggccgcgata tgtacgtgga ccaggagctg
     9601 gacatcaacc gcctgtccga ctacgatgtg gaccatatcg tgccgcagag ctttctgaag
     9661 gatgacagca tcgataacaa ggtgctgacc cgcagcgata agaaccgcgg caagtccgat
     9721 aacgtgccct cggaggaggt cgtgaagaag atgaagaact attggcgcca gctgctgaac
     9781 gccaagctga ttacccagcg caagttcgac aatctgacca aggccgagcg cggaggcctg
     9841 agcgagctgg ataaggccgg cttcatcaag cgccagctgg tggaaacccg ccagattacc
     9901 aagcacgtgg cccagattct ggatagccgc atgaacacga agtacgacga gaatgataag
     9961 ctgattcgcg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccgcaag
    10021 gatttccagt tctataaagt gcgcgagatc aacaactacc accacgccca cgatgcctac
    10081 ctgaacgccg tcgtgggaac cgccctgatt aagaagtatc ccaagctgga gagcgagttc
    10141 gtgtacggcg attataaggt gtacgacgtg cgcaagatga tcgccaagtc cgagcaggag
    10201 atcggcaagg ccaccgccaa gtactttttc tacagcaaca tcatgaattt cttcaagacc
    10261 gagattaccc tggccaacgg cgagatccgc aagcgcccac tgatcgaaac gaatggcgaa
    10321 accggcgaga ttgtgtggga caagggccgc gattttgcca ccgtgcgcaa ggtgctgtcc
    10381 atgccccaag tgaacattgt gaaaaagacc gaggtgcaga cgggcggctt cagcaaggag
    10441 tccattctgc cgaagcgcaa cagcgacaag ctgatcgccc gcaagaagga ttgggacccc
    10501 aagaagtacg gcggcttcga tagccccacc gtggcctata gtgtgctggt ggtggccaag
    10561 gtggagaagg gcaagtccaa gaagctgaag tccgtgaagg agctgctggg catcaccatc
    10621 atggagcgca gcagctttga gaagaaccca atcgatttcc tggaggccaa gggctacaag
    10681 gaagtgaaga aggatctgat catcaagctg ccgaagtact ccctgttcga gctggagaac
    10741 ggacgcaagc gcatgctggc cagtgccggc gagctgcaga agggaaacga gctggccctg
    10801 ccctcgaaat atgtgaactt cctgtacctg gccagccact acgagaagct gaagggcagc
    10861 cccgaggata acgagcagaa gcagctgttt gtggagcagc acaagcacta cctggacgag
    10921 atcattgagc agatcagcga gttcagcaag cgcgtgatcc tggccgacgc caatctggat
    10981 aaggtgctga gcgcctacaa caagcaccgc gacaagccaa tccgcgagca ggccgagaac
    11041 atcatccacc tgttcaccct gaccaacctg ggagccccag ccgccttcaa gtacttcgat
    11101 accaccatcg atcgcaagcg ctataccagc accaaggagg tgctggacgc caccctgatc
    11161 caccagagca tcaccggcct gtacgaaacc cgcatcgatc tgtcgcagct gggaggcgat
    11221 aagcgcccag ccgccaccaa gaaggccgga caggccaaaa agaagaagta gctcgaggaa
    11281 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
    11341 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
    11401 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
    11461 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc
    11521 cgcaggaacc cctagtgata agggcgaatt cgacccagct ttcttgtaca aagtggtgat
    11581 atccggtggc ggaggcagtg gaaagtctta cccaactgtg agtgctgatt accaggacgc
    11641 cgttgagaag gcgaagaaga agctcagagg cttcatcgct gagaagagat gcgctcctct
    11701 aatgctccgt ttggcattcc actctgctgg aacctttgac aagggcacga agaccggtgg
    11761 acccttcgga accatcaagc accctgccga actggctcac agcgctaaca acggtcttga
    11821 catcgctgtt aggcttttgg agccactcaa ggcggagttc cctattttga gctacgccga
    11881 tttctaccag ttggctggcg ttgttgccgt tgaggtcacg ggtggaccta aggttccatt
    11941 ccaccctgga agagaggaca agcctgagcc accaccagag ggtcgcttgc ccgatcccac
    12001 taagggttct gaccatttga gagatgtgtt tggcaaagct atggggctta ctgaccaaga
    12061 tatcgttgct ctatctgggg gtcacactat tggagctgca cacaaggagc gttctggatt
    12121 tgagggtccc tggacctcta atcctcttat tttcgacaac tcatacttca cggagttgtt
    12181 gagtggtgag aaggaaggtc tccttcagct accttctgac aaggctcttt tgtctgaccc
    12241 tgtattccgc cctctcgttg acaaatatgc agcggacgaa gatgccttct ttgctgatta
    12301 cgctgaggct caccaaaagc tttccgagct tgggtttgct gatgccagcg gaggcggtgg
    12361 ctccgaacag aaacttatca gcgaggagga tctcggcgag cagaagctga tttcggaaga
    12421 agaccttgga gagcaaaagc tcatatccga agaggacctg tcgtaagaat tcctgcagcc
    12481 cgggggatcc actagttcta gtcgaccatg aagatcaaga tcattgcccc gccagagcgc
    12541 aagtactctg tctggatcgg tggctccatc ctggcttcgc tgtccacctt ccagcagatg
    12601 tggatctcca agcaggagta cgacgagtcc ggcccctcca ttgtgcaccg caagtgcttc
    12661 taagaaggat cgcttgtctg ggcaagagga tcaggatcgg gatggtcttg attctgctgg
    12721 aggaggagga ggagaagtcg aggaagcagc agcgaaagtg caagtgcgag tggtggaagt
    12781 ttggagtgca gcacaacaaa atcaacaaca acaccaacta caagatgaaa agagcggaac
    12841 cacctgcaca ccatcatcac tatcatcatc gttttgggcg catgttgtgt ggttccagcg
    12901 tattaatata attaatttat tccacatgag atatgatatg atatactatg tattttttgt
    12961 ttttttttta tttgtaaacc tttaatataa caagaactac aaaaaatgaa aatgagcgaa
    13021 aatgcatatt ctgccattcc acacacacac caacaacacc caacacacgc acacccacaa
    13081 gcttacacac acacattcgc ggcatgacaa ggacatcaag ataaagaaga acttaaagaa
    13141 gatatttccc aaagcgcaaa aagaacacac acacattgca aaacacaaac aacacactag
    13201 cgttttgtac aattcgtcag caaccttatg tattattttt taattatgat gtaattataa
    13261 acaaagtgaa acaaaaatat gaaaacaaaa aggaaaatca aatctgtctt ctctttctcc
    13321 cgctctcctc gctctctgct gctaacctcg ccctctcctc tctcatcttt ttgtctgtct
    13381 ctcttccaca tttttgccgg ccggcaaaat aataacccac acacactcac acttggctgc
    13441 agtttcgcgt gcgatattca cacacattca agcatacaag catacataca tatgtatttt
    13501 ttttttattt gtacactttt ctaattgcat gcgtatcgat tgataagttt acgcctgaaa
    13561 atgttaatta aaatgtgaaa atgcaactga aaaactgatg aaatgaaaca acaacaagcg
    13621 aacaatttgc tacatgtgta ttgtctaaca accgttactg caacggttgc ttcggaaaag
    13681 ggtgaagagg aagagggcac gaggtcgact agagcggccg ccgacgcgag gctggatggc
    13741 cttccccatt atgattcttc tcgcttccgg cggcatcggg atg
//
