LOCUS       sequence-292191-       14423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CaCas9/gRNA Solo entry plasmid for cloning guides - contains stuffer
            with BsmBI sites - inserts at Neut5L - Recyclable for serial 
            mutagenesis (Mal2-FLP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14423)
  AUTHORS   Vyas VK, Bushkin GG, Bernstein DA, Getz MA, Sewastianik M, Barrasa 
            MI, Bartel DP, Fink GR
  TITLE     New CRISPR Mutagenesis Strategies Reveal Variation in Repair 
            Mechanisms among Fungi.
  JOURNAL   mSphere. 2018 Apr 25;3(2). pii: 3/2/e00154-18. doi: 
            10.1128/mSphere.00154-18. Print 2018 Apr 25.
  PUBMED    29695624
REFERENCE   2  (bases 1 to 14423)
  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: "mSphere."; 
            date: "2018-04-25"; volume: "3(2). pii: 3/2/e00154-18. doi"; pages: 
            " 10.1128/mSphere.00154-18. Print 2018 Apr 25"
COMMENT     Sequence Label: pV1524
FEATURES             Location/Qualifiers
     source          1..14423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             15..4118
                     /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             15..4118
                     /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             4131..4151
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4155..4175
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4179..4199
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4200..4265
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      4330..4577
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4346..4364)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             5137..6408
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=FLP
                     /note="FLP is a site-specific recombinase from 
                     Saccharomyces cerevisiae. Recombination occurs at FRT 
                     sequences."
                     /translation="MPQFDILCKTPLKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     terminator      complement(6835..7032)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(7633..7976)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     misc_RNA        9045..9120
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(9200..9233)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     promoter        complement(9600..9618)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9639..9655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9639..9655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9652..9674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9663..9679
                     /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(9687..9693,9694..9711,9712..9717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9732..9753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9870..9887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10041..10629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10121..10140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10800..11591,11592..11660))
                     /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     11423..11442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11661..11765)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11791..12246)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11928..11949)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12140..12159
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12373..12395
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     12387..12404
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     12388..12404
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     12414..12433
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        12414..12432
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(12885..12918)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
ORIGIN
        1 tattacaacc cgggatggat aaaaagtata gtattggttt agatattggt actaactctg
       61 tgggttgggc agttatcact gacgaatata aagttccatc aaagaaattt aaggtgttag
      121 gtaacactga cagacactca ataaaaaaga atcttatcgg tgctcttttg ttcgactccg
      181 gtgaaactgc cgaggctaca cgtttaaaaa gaacagcaag aagaagatat acccgtagaa
      241 aaaatagaat atgttattta caagaaatct tttctaatga aatggctaaa gttgatgatt
      301 cctttttcca tagattggaa gagtcatttt tggttgaaga agacaaaaag catgagagac
      361 atccaatctt tgggaatata gttgatgaag tggcttacca tgaaaaatat cctaccattt
      421 atcatttaag aaagaaattg gtagattcaa ctgataaagc tgaccttaga ttaatctatt
      481 tagcacttgc ccatatgatt aaatttagag gtcatttttt gattgaaggt gatttgaacc
      541 cagataattc tgacgtggat aaattattta ttcaattagt ccaaacctac aaccaattat
      601 ttgaggaaaa tccaattaat gctagtggtg tcgatgccaa agctatatta tcagccagat
      661 tatcaaaatc tagacgtttg gaaaatttga ttgcccaatt gccaggagaa aaaaagaatg
      721 gattatttgg aaacttgatc gcattatcat tgggtttgac accaaatttt aaatctaatt
      781 ttgatttagc tgaagatgct aaattacaat tatcaaaaga cacctatgac gacgatttgg
      841 acaatttact tgctcaaatt ggtgatcaat atgcagattt gttcttagct gctaaaaact
      901 tatctgatgc tattttgttg tctgatattt tgagagtgaa cacagaaata accaaagctc
      961 cattatcagc atctatgatc aaacgttatg atgaacacca tcaggatttg actttattga
     1021 aagctttggt gagacaacaa ttgccagaga agtataaaga aatctttttc gatcaatcta
     1081 aaaacgggta tgcaggttat attgatgggg gtgcctccca agaggaattt tacaaattta
     1141 taaaacctat tttagaaaag atggatggga ctgaggaact tttggtcaaa ttgaacagag
     1201 aagatttgtt acgtaaacag agaacttttg ataatggtag tatacctcac caaattcatt
     1261 tgggtgagtt gcatgcaatt ttaagaagac aagaagattt ttatccattt ttaaaagata
     1321 atagagaaaa aatcgagaaa attttaacct ttagaattcc atactatgtt gggcctttgg
     1381 ctagaggtaa ttcaagattt gcctggatga cacgtaaatc agaagaaact attacccctt
     1441 ggaattttga agaggttgtt gataaaggag catcagcaca gagttttatt gaaagaatga
     1501 ccaatttcga taaaaactta ccaaatgaaa aagttttacc aaaacattcc ttgttatacg
     1561 aatattttac tgtttacaat gaacttacaa aggttaaata tgttactgaa ggtatgcgta
     1621 agccagcctt tttatctgga gaacagaaaa aggcaatagt tgatttattg tttaaaacaa
     1681 atagaaaagt tactgttaaa caattaaaag aagattactt taagaaaatt gaatgttttg
     1741 attcagttga aatcagtggt gttgaagaca gatttaatgc tagtttagga acttaccatg
     1801 atttacttaa aattatcaaa gataaagatt tcttggataa cgaagaaaat gaagacattt
     1861 tagaagacat tgttttaacc ttaactttat tcgaagatag agagatgatt gaagaacgtt
     1921 tgaagactta tgcacatttg tttgacgata aagtgatgaa acagttgaaa agaagacgtt
     1981 atactggatg gggtagattg tctcgtaaat tgatcaatgg aattagagat aaacaaagtg
     2041 gtaaaactat cttggacttt ttgaaatctg acggatttgc taatagaaat ttcatgcaat
     2101 tgatccacga cgatagtttg acatttaaag aagacatcca aaaggcccaa gtgagtgggc
     2161 aaggtgattc attacatgaa catattgcaa atttagccgg atctcctgct attaagaaag
     2221 ggatattaca aactgttaaa gttgtggatg aattagtgaa agtaatggga agacataaac
     2281 ctgaaaacat tgtcattgag atggcaagag aaaatcaaac tacacaaaaa ggacagaaaa
     2341 atagtagaga acgtatgaaa agaatagaag agggtattaa agaattgggt agtcaaatat
     2401 tgaaagaaca cccagtggaa aatacccagt tgcaaaatga aaaattatat ctttactacc
     2461 ttcaaaatgg acgtgatatg tatgttgatc aggaattaga tataaataga ctttcagatt
     2521 atgatgtaga tcatatagtt ccacaatctt tcttgaaaga tgattccata gacaataaag
     2581 tattaactag aagtgataaa aatagaggta aaagtgataa tgtcccaagt gaggaagtcg
     2641 tcaaaaagat gaaaaattac tggcgtcaac ttttgaatgc taaattaatt actcaaagaa
     2701 aatttgataa tttgactaaa gcagaaagag gtgggctttc tgaattagat aaagccgggt
     2761 tcattaaaag acaattggtc gaaactagac aaattactaa acatgttgcc caaattttag
     2821 attcccgtat gaacactaag tatgacgaaa atgataagtt aatacgtgag gttaaagtca
     2881 ttactttaaa atcaaaactt gtctctgatt tcagaaagga tttccaattc tataaagtta
     2941 gagaaattaa taattatcat catgctcatg atgcatattt gaatgctgta gttggaactg
     3001 ctttaatcaa gaaataccct aaattagaat ctgaatttgt atatggtgat tacaaagtct
     3061 atgatgttag aaagatgatt gctaaatcag aacaagaaat tggtaaagct acagctaaat
     3121 acttctttta ctctaacatt atgaatttct ttaaaacaga aattactttg gcaaacggtg
     3181 aaattagaaa aagacctctt attgaaacaa atggtgagac tggagagata gtttgggaca
     3241 aagggcgtga tttcgctact gttagaaaag ttttatcaat gccacaagtt aacattgtaa
     3301 agaaaacaga ggttcaaact ggtggtttct caaaagaaag tattttgcct aaaagaaata
     3361 gtgataaatt aattgccaga aaaaaggatt gggatccaaa gaaatatggt ggtttcgact
     3421 caccaaccgt agcctattct gttttggttg tggcaaaggt tgaaaagggt aaaagtaaaa
     3481 agcttaaatc agtaaaagaa cttttgggta ttacaataat ggaaagaagt tcctttgaaa
     3541 agaaccctat tgattttttg gaagctaaag gttataagga agtaaagaag gacttaataa
     3601 tcaaattgcc taaatattct ttatttgaat tagaaaatgg gagaaaaaga atgttggctt
     3661 ctgctggaga attgcaaaag ggtaatgaat tagcattgcc ttccaaatat gttaacttct
     3721 tgtatttagc ttcacactat gaaaagttga aagggtcacc agaagataac gagcaaaaac
     3781 aattatttgt tgaacaacac aaacactact tagatgagat tatagaacaa attagtgaat
     3841 tcagtaaaag agtgatatta gctgatgcaa atttagataa agttttgtca gcctataaca
     3901 aacatagaga taagccaatt agagaacaag cagaaaacat tattcactta tttaccctta
     3961 ccaatttagg agcacctgct gctttcaagt attttgatac aacaattgat cgtaaaagat
     4021 atacctcaac aaaagaagtc ttagacgcca ccttaattca tcaatcaatc actggattgt
     4081 atgagacaag aattgatttg tctcaattgg gtggtgatga aggggctgat cctaagaaga
     4141 aaagaaaagt tgatccaaag aaaaagcgta aggtggatcc taagaaaaag agaaaggttg
     4201 actacaaaga ccatgacggt gattataaag atcatgacat cgactacaag gatgacgatg
     4261 acaagtgata atgactgcag agatccatcg acctgccgcc aagctaattc cgggcgaatt
     4321 tctgtcgagt catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg
     4381 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta
     4441 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca
     4501 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct
     4561 cgaaggcttt aatttgcggc cgggcccccc cgtttgatat ttttgtctag taccatctgt
     4621 accattacac ttaaattatc tttatatctg tctaactcga ctgtctggat ttcattgatg
     4681 tagtcgtatg catcgttagt tccaaaaaat attgtcatca atttgatatt ggtttccgac
     4741 tctaaaattt ttggaagaat ttgtctagcg tgctctgagt tgtagccact gaaaccacgg
     4801 ttaataacat ccaattttcg gatatacaca ttctgtaatg ctggatgaaa gccatactgg
     4861 gtacaactaa actgggtgat ggagtcaccg aacaacacaa atttaccgta ttccatgatt
     4921 gctatggttg agaatttttt ttttttcttg tcccacgcca tttttcaaat tatgcagttg
     4981 agaatgttag tttttgtgta caccccgttc gctgaatatt tcggaataat tcaaagattg
     5041 gggagtgggg gaggcgatag acgaagacac ggtataaaaa tgggcaaaat tttccccaac
     5101 tttttgcagt ggtttaacta ataatcaact acaatgatgc cacaatttga tatattatgt
     5161 aaaacaccac ttaaggtgct tgttcgtcag tttgtggaaa ggtttgaaag accttcaggt
     5221 gagaaaatag cattatgtgc tgctgaacta acctatttat gttggatgat tacacataac
     5281 ggaacagcaa tcaagagagc cacattcatg agctataata ctatcataag caattcgttg
     5341 agtttcgata ttgtcaataa atcactccag tttaaataca agacgcaaaa agcaacaatt
     5401 ttggaagcct cattaaagaa attgattcct gcttgggaat ttacaattat tccttactat
     5461 ggacaaaaac atcaatctga tatcactgat attgtaagta gtttgcaatt acagttcgaa
     5521 tcatcggaag aagcagataa ggggaatagc cacagtaaaa aaatgcttaa agcacttcta
     5581 agtgagggtg aaagcatctg ggagatcact gagaaaatac taaattcgtt tgagtatact
     5641 tcgagattta caaaaacaaa aactttatac caattcctct tcctagctac tttcatcaac
     5701 tgtggaagat tcagcgatat taagaacgtt gatccgaaat catttaaatt agtccaaaat
     5761 aagtatttgg gagtaataat ccagtgttta gtgacagaga caaagacaag cgttagtagg
     5821 cacatatact tctttagcgc aaggggtagg atcgatccac ttgtatattt ggatgaattt
     5881 ttgaggaatt ctgaaccagt cctaaaacga gtaaatagga ccggcaattc ttcaagcaat
     5941 aaacaggaat accaattatt aaaagataac ttagtcagat cgtacaataa agctttgaag
     6001 aaaaatgcgc cttattcaat ctttgctata aaaaatggcc caaaatctca cattggaaga
     6061 catttgatga cctcatttct ttcaatgaag ggcctaacgg agttgactaa tgttgtggga
     6121 aattggagcg ataagcgtgc ttctgccgtg gccaggacaa cgtatactca tcagataaca
     6181 gcaatacctg atcactactt cgcactagtt tctcggtact atgcatatga tccaatatca
     6241 aaggaaatga tagcattgaa ggatgagact aatccaattg aggagtggca gcatatagaa
     6301 cagctaaagg gtagtgctga aggaagcata cgataccccg catggaatgg gataatatca
     6361 caggaggtac tagactacct ttcatcctac ataaatagac gcatataaga gtgaaattct
     6421 ggaaatctgg aaatctggtt ttgtattctt gttattcttc tttttgttat tacatatata
     6481 acttgttact tttttaaaaa aatctttgta tattttataa atatataaaa ctaaatttaa
     6541 gaaaaagaga aaaatgtttt atttgagaga ttgaaatttt acttgaattt agcttagctt
     6601 ttataaagta ttattatgta aaaaaacaaa acaaatatac atcaaaaagt taagactata
     6661 aaatagccac ccaaggcatt tctatatctt gttgttgttg ttttcatctt ctgtatcaga
     6721 ggaacttatt ttattatttt cgtcacgggt attttctctt gtttgatgat tcatcccatt
     6781 cattccatca taaaatgtcg acactggatg gcggcgttag tatcgaatcg acagcagtat
     6841 agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc
     6901 atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa
     6961 tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt
     7021 tttattgtca gtactgactc gagttattat ggacatggca tagacatata caaagcttgt
     7081 tcaccatcgg aagcagtacc atcgtataaa gcagtatcca aaccacacaa agtgaaaccc
     7141 attcctctat aagcatgaat agctggagca ttaacattgg taacttccaa ccacaaatga
     7201 ccagcacctc tttctctggc gaattcagta gccaaaccca tcaaagctct accaacacca
     7261 tgacctctat gttctggagc aacttcaata tcttcaacag tcaatcttct gttccaacca
     7321 gaataagaaa caacaacgaa accagccaaa tcaccatcat caccataagc aacgaaagtt
     7381 ctagaatctg gatcaccatc ttcaccagca tcggattcat catcggattc atcatctggg
     7441 aaaaccttag tcaatggtgg atcaactgga acttctctca aagtgaaacc atcaccagta
     7501 gcagtaactc taaaaacagt atcggtagtg aaagaaccat ccaaagcttc aatagcttca
     7561 gcatcacctg gaacagaagt tctgtatcta taagcagtat catccaaagt agtagacata
     7621 attgtaggat ccggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt
     7681 ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaac cctaaccttt tatatttctc
     7741 tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg
     7801 caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg
     7861 gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat
     7921 gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg
     7981 gccgggtgac ccggcgggga cgaggcaagc ttgatgtgcg gccgcaagtg attagactta
     8041 gtccgttcaa atcaagcaca actctgttca ttgtttcaac aagaattaat tcaaaaacag
     8101 gttcggtgca taatttgcaa aaaaatattg cagcttctgt ggctcgaaca cagtacctcc
     8161 agatttcagg tttgaaatac ttcagtctga cgctctccca gatgagctaa agctgcaata
     8221 agaaaaccca cgccgggatt cgaacccgga atcctttgat tagaagtcaa aagcgataac
     8281 catttcgcca cgcaggccta cttgatgggt ttgtaaatgg tctacttttt cagacctaac
     8341 agaaatttta atgaaagtca tattcttata caataaaact gtgtcataaa agcagatatt
     8401 cgactttcgt agattatata ggacccaaga actaaaattt aatgccatat tatgcatttt
     8461 taatctgtaa aagtgttgtt tccaacctat cacaagtacg ttcttgtaac ttgtgtttgt
     8521 agggttgcaa atgaatcata acaacatctc aacagaacat gtatagcaaa gcttagtata
     8581 aaatcagtgt tttgagaggc aatccaagaa tgtttacatc aaagtttcaa taaatatcga
     8641 ccgaaactga aaatcttttt aggttattgt tcactttttt gtaaatattt aaacattttt
     8701 tggacctaaa aaaatacaag caccaattac gtaccaagaa gcatctaatc aactcccaga
     8761 tcaccactat acatttaaaa gtcattggtc aataactata ctcgagtatt gcctcatcaa
     8821 agaaacaatc aaatattata gatactcact ccatcacgtg ataatttcac tggtatggaa
     8881 aagtggaaaa ttttataaaa aaaaatttga tgcctttggc atagctgaaa cttcggccca
     8941 ataggattgg agaatatgtt ttcgcagcgt tcttacaatt aaattgtggt ggaagttcga
     9001 gacttgcgta aactattttt aatttggaga cggaattccg tctcgtttta gagctagaaa
     9061 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     9121 tttttttctc gagttttttt atcgagtgtt taaggataat gataactgaa gagaagaatt
     9181 agttttgccg ccaccgcggg aagttcctat actttctaga gaataggaac ttcacgccgg
     9241 taaacaagtg gtattcaagc acaattcagt tttctggatc tctaataatt gcaattgcaa
     9301 ttgcttcaca tactctgttt ataattcagt ttctttctct tgatgttact gatatattaa
     9361 gttaataaat accaataact ttttttcaaa aatcttctat tttcctttca attgattatt
     9421 taaacacgaa tcgttaataa gctgtgattg ctttttcttt ttctttttct tttctttgtt
     9481 caagtttgaa aaaagtaaaa aaaaaggttt ctcttgttcg atgtagaatt cagtgggtta
     9541 tagttgtaat gatatatgga ataaaaagaa agaaagaaag agagctccag cttttgttcc
     9601 ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt tcctgtgtga
     9661 aattgttatc cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc
     9721 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     9781 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     9841 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     9901 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     9961 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
    10021 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
    10081 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
    10141 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
    10201 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
    10261 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
    10321 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
    10381 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
    10441 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
    10501 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
    10561 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
    10621 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
    10681 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
    10741 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
    10801 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
    10861 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
    10921 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
    10981 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
    11041 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
    11101 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
    11161 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
    11221 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
    11281 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
    11341 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
    11401 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
    11461 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
    11521 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
    11581 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
    11641 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
    11701 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
    11761 ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat
    11821 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
    11881 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
    11941 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
    12001 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
    12061 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
    12121 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
    12181 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
    12241 gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
    12301 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
    12361 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg
    12421 actcactata gggcgaattg aggcctgcat gcggtaccat gaagaatgct gaatcacttg
    12481 ataggattta gttccattat ggtactaaga agtaatatta cttctatgaa aaggttgaat
    12541 ccgagatcaa aattcatctt ttcatatact ctggagaaaa aaaaaaaagt aattgtagta
    12601 agaatgacaa gtatcagttg agaaaaaaag taacaaagag aaagctcgga ggaggctccc
    12661 caaaggtttt atcaccaatg gtggaaccac taacccaaga acagaaaaag cattgatatt
    12721 ttgaaaaaat acaatactca tcttttaacc cgaacctttt cttgtccttt aaaactttta
    12781 tctcatatct gtccaaagtc atattgtctt actactttct ctccttcatc gtccttcctg
    12841 ccactactac cactgggagt ttcgttcttc tcgatacgga tcctgaagtt cctatacttt
    12901 ctagagaata ggaacttcat tagctttact tcctgcacta gcagtggttg gatcaacaga
    12961 atcttcataa tcatcaaaat cgtcttttga agaccccccg tttgatgtat ggccctgtct
    13021 tttcatcaaa ctttttatat agttgactga actgaggcta aatatgtgat catcttcact
    13081 atagacaatc tttctcttat ttgcaccacc gccaccacca gtctttgaga aattctcaaa
    13141 accttttacg atattaccaa gcgggctctc ttcgaaataa tctatctctt tttgatatat
    13201 cgaatcctct agcgtggtta gctttctagt tagttcttgc ttcttaagaa tttgctggat
    13261 tagtttattt ttcaattcaa cgtatttctc agagtcatct ttagattttg atgaagatgt
    13321 gcgttcattc gctatatcct tcttggtcgt gtcttttcga tcctccttgg ctggcactga
    13381 actcgtcttt tttggcgttg ctgttccaga cagacttatc tcattagatt tggaacttgt
    13441 gggtttaaca tcatttgcat ctttagtaga catgattgtg taataccgtg attgtttgtt
    13501 ttgaaaggtc tgtcatattt ctatcaattt caaaacaaaa tgttcatcag aaaaaagcca
    13561 aaaatgtctc ttctagcttc ttagtggtgt cgcaaaatac acaatgtcgc tcaacaatcc
    13621 acattcccgg cgcatagctc aaatcacatg actacagcta acaattacac aaaaaaattc
    13681 tcttttcgat gtagcaacta tcttcaacta aaacattttc tccttcggcc catgattgtc
    13741 ctccgggtcg acagcaagcc gttacaattg agatggaaag cgacctacct tcactcgata
    13801 aggtgcttaa ttgtacttca tataaatctg gcccggatct aaacaaatga gttccattaa
    13861 gccgtgggtt ctcaattagg gtttttgttt ttgatttaga aaaaagagat caagatttgt
    13921 ttacaggtga tgcctttttt tagaacttat gcgttgcaaa agttgactaa cgatttctat
    13981 aaggtgatcc acactaatta tacaaacgta caaacagaca tacttttcct gcgttcacct
    14041 gatgttggcc agatttctct cttcattgca tagaacataa ccacactagg gcaacagaaa
    14101 aaaaaaaaaa aagtgcatcg ggaagttgtg ttccattcat tatatgtcta ctactgcata
    14161 tgagtagccc acccaccacc accatagtaa gtttttgtgt atgcgcgccg tcaggttatt
    14221 tcatttctga atttttcaac caccttactc cctttattgt tgattgacaa ttttgctcac
    14281 agtaagatct tttagactcc aattaatata aaataagtct gattttccaa ttcctgtttt
    14341 ttcttttttt ttctgtttct atttctttcc ttttctccct tttttttaat tcttcattca
    14401 atcatcaatt gataattcag gaa
//
