LOCUS       sequence-212464-       16324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CaCas9/gRNA Solo entry plasmid for cloning guides - contains stuffer
            with BsmBI sites - integrates at ENO1 - Recyclable for serial 
            mutagenesis (Sap2-FLP).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16324)
  AUTHORS   Vyas VK, Barrasa MI, Fink GR
  TITLE     A CRISPR system permits genetic engineering of essential genes and 
            gene families.
  JOURNAL   Sci Adv. 2015;1(3):e1500248.
  PUBMED    25977940
REFERENCE   2  (bases 1 to 16324)
  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: "Sci Adv."; 
            date: "2015"; volume: "1(3)"; pages: "e1500248"
COMMENT     Sequence Label: pV1200
FEATURES             Location/Qualifiers
     source          1..16324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(18..473)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(155..176)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     367..386
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     600..622
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     614..631
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     615..631
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     641..660
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        641..659
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2245..6348
                     /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             2245..6348
                     /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             6361..6381
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6385..6405
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6409..6429
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6430..6495
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      6560..6807
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6576..6594)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    complement(6827..6860)
                     /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)"
     primer_bind     complement(6862..6878)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6862..6878)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             7957..9228
                     /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="MPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
                     THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
                     IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
                     SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
                     KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
                     SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
                     TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
                     YPAWNGIISQEVLDYLSSYINRRI"
     terminator      complement(9655..9852)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        complement(10453..10796)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     misc_RNA        11864..11939
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    12019..12052
                     /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)"
     protein_bind    complement(12059..12092)
                     /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)"
     primer_bind     complement(14151..14171)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(14151..14169)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(14190..14206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14190..14206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14203..14225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    14214..14230
                     /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(14238..14244,14245..14262,14263..14268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    14283..14304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(14421..14438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(14592..15180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(14672..14691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(15351..16142,16143..16211))
                     /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     15974..15993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(16212..16316)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
       61 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
      121 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
      181 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
      241 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
      301 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
      361 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
      421 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
      481 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
      541 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
      601 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg
      661 cgaattgagg cctgcatgcg gtacctgcca ctactaccac tgggagtttc gttcttctcg
      721 atactattag ctttacttcc tgcactagca gtggttggat caacagaatc ttcataatca
      781 tcaaaatcgt cttttgaaga ccccccgttt gatgtatggc cctgtctttt catcaaactt
      841 tttatatagt tgactgaact gaggctaaat atgtgatcat cttcactata gacaatcttt
      901 ctcttatttg caccaccgcc accaccagtc tttgagaaat tctcaaaacc ttttacgata
      961 ttaccaagcg ggctctcttc gaaataatct atctcttttt gatatatcga atcctctagc
     1021 gtggttagct ttctagttag ttcttgcttc ttaagaattt gctggattag tttatttttc
     1081 aattcaacgt atttctcaga gtcatcttta gattttgatg aagatgtgcg ttcattcgct
     1141 atatccttct tggtcgtgtc ttttcgatcc tccttggctg gcactgaact cgtctttttt
     1201 ggcgttgctg ttccagacag acttatctca ttagatttgg aacttgtggg tttaacatca
     1261 tttgcatctt tagtagacat gattgtgtaa taccgtgatt gtttgttttg aaaggtctgt
     1321 catatttcta tcaatttcaa aacaaaatgt tcatcagaaa aaagccaaaa atgtctcttc
     1381 tagcttctta gtggtgtcgc aaaatacaca atgtcgctca acaatccaca ttcccggcgc
     1441 atagctcaaa tcacatgact acagctaaca attacacaaa aaaattctct tttcgatgta
     1501 gcaactatct tcaactaaaa cattttctcc ttcggcccat gattgtcctc cgggtcgaca
     1561 gcaagccgtt acaattgaga tggaaagcga cctaccttca ctcgataagg tgcttaattg
     1621 tacttcatat aaatctggcc cggatctaaa caaatgagtt ccattaagcc gtgggttctc
     1681 aattagggtt tttgtttttg atttagaaaa aagagatcaa gatttgttta caggtgatgc
     1741 ctttttttag aacttatgcg ttgcaaaagt tgactaacga tttctataag gtgatccaca
     1801 ctaattatac aaacgtacaa acagacatac ttttcctgcg ttcacctgat gttggccaga
     1861 tttctctctt cattgcatag aacataacca cactagggca acagaaaaaa aaaaaaaaag
     1921 tgcatcggga agttgtgttc cattcattat atgtctacta ctgcatatga gtagcccacc
     1981 caccaccacc atagtaagtt tttgtgtatg cgcgccgtca ggttatttca tttctgaatt
     2041 tttcaaccac cttactccct ttattgttga ttgacaattt tgctcacagt aagatctttt
     2101 agactccaat taatataaaa taagtctgat tttccaattc ctgttttttc tttttttttc
     2161 tgtttctatt tctttccttt tctccctttt ttttaattct tcattcaatc atcaattgat
     2221 aattcaggaa tattacaacc cgggatggat aaaaagtata gtattggttt agatattggt
     2281 actaactctg tgggttgggc agttatcact gacgaatata aagttccatc aaagaaattt
     2341 aaggtgttag gtaacactga cagacactca ataaaaaaga atcttatcgg tgctcttttg
     2401 ttcgactccg gtgaaactgc cgaggctaca cgtttaaaaa gaacagcaag aagaagatat
     2461 acccgtagaa aaaatagaat atgttattta caagaaatct tttctaatga aatggctaaa
     2521 gttgatgatt cctttttcca tagattggaa gagtcatttt tggttgaaga agacaaaaag
     2581 catgagagac atccaatctt tgggaatata gttgatgaag tggcttacca tgaaaaatat
     2641 cctaccattt atcatttaag aaagaaattg gtagattcaa ctgataaagc tgaccttaga
     2701 ttaatctatt tagcacttgc ccatatgatt aaatttagag gtcatttttt gattgaaggt
     2761 gatttgaacc cagataattc tgacgtggat aaattattta ttcaattagt ccaaacctac
     2821 aaccaattat ttgaggaaaa tccaattaat gctagtggtg tcgatgccaa agctatatta
     2881 tcagccagat tatcaaaatc tagacgtttg gaaaatttga ttgcccaatt gccaggagaa
     2941 aaaaagaatg gattatttgg aaacttgatc gcattatcat tgggtttgac accaaatttt
     3001 aaatctaatt ttgatttagc tgaagatgct aaattacaat tatcaaaaga cacctatgac
     3061 gacgatttgg acaatttact tgctcaaatt ggtgatcaat atgcagattt gttcttagct
     3121 gctaaaaact tatctgatgc tattttgttg tctgatattt tgagagtgaa cacagaaata
     3181 accaaagctc cattatcagc atctatgatc aaacgttatg atgaacacca tcaggatttg
     3241 actttattga aagctttggt gagacaacaa ttgccagaga agtataaaga aatctttttc
     3301 gatcaatcta aaaacgggta tgcaggttat attgatgggg gtgcctccca agaggaattt
     3361 tacaaattta taaaacctat tttagaaaag atggatggga ctgaggaact tttggtcaaa
     3421 ttgaacagag aagatttgtt acgtaaacag agaacttttg ataatggtag tatacctcac
     3481 caaattcatt tgggtgagtt gcatgcaatt ttaagaagac aagaagattt ttatccattt
     3541 ttaaaagata atagagaaaa aatcgagaaa attttaacct ttagaattcc atactatgtt
     3601 gggcctttgg ctagaggtaa ttcaagattt gcctggatga cacgtaaatc agaagaaact
     3661 attacccctt ggaattttga agaggttgtt gataaaggag catcagcaca gagttttatt
     3721 gaaagaatga ccaatttcga taaaaactta ccaaatgaaa aagttttacc aaaacattcc
     3781 ttgttatacg aatattttac tgtttacaat gaacttacaa aggttaaata tgttactgaa
     3841 ggtatgcgta agccagcctt tttatctgga gaacagaaaa aggcaatagt tgatttattg
     3901 tttaaaacaa atagaaaagt tactgttaaa caattaaaag aagattactt taagaaaatt
     3961 gaatgttttg attcagttga aatcagtggt gttgaagaca gatttaatgc tagtttagga
     4021 acttaccatg atttacttaa aattatcaaa gataaagatt tcttggataa cgaagaaaat
     4081 gaagacattt tagaagacat tgttttaacc ttaactttat tcgaagatag agagatgatt
     4141 gaagaacgtt tgaagactta tgcacatttg tttgacgata aagtgatgaa acagttgaaa
     4201 agaagacgtt atactggatg gggtagattg tctcgtaaat tgatcaatgg aattagagat
     4261 aaacaaagtg gtaaaactat cttggacttt ttgaaatctg acggatttgc taatagaaat
     4321 ttcatgcaat tgatccacga cgatagtttg acatttaaag aagacatcca aaaggcccaa
     4381 gtgagtgggc aaggtgattc attacatgaa catattgcaa atttagccgg atctcctgct
     4441 attaagaaag ggatattaca aactgttaaa gttgtggatg aattagtgaa agtaatggga
     4501 agacataaac ctgaaaacat tgtcattgag atggcaagag aaaatcaaac tacacaaaaa
     4561 ggacagaaaa atagtagaga acgtatgaaa agaatagaag agggtattaa agaattgggt
     4621 agtcaaatat tgaaagaaca cccagtggaa aatacccagt tgcaaaatga aaaattatat
     4681 ctttactacc ttcaaaatgg acgtgatatg tatgttgatc aggaattaga tataaataga
     4741 ctttcagatt atgatgtaga tcatatagtt ccacaatctt tcttgaaaga tgattccata
     4801 gacaataaag tattaactag aagtgataaa aatagaggta aaagtgataa tgtcccaagt
     4861 gaggaagtcg tcaaaaagat gaaaaattac tggcgtcaac ttttgaatgc taaattaatt
     4921 actcaaagaa aatttgataa tttgactaaa gcagaaagag gtgggctttc tgaattagat
     4981 aaagccgggt tcattaaaag acaattggtc gaaactagac aaattactaa acatgttgcc
     5041 caaattttag attcccgtat gaacactaag tatgacgaaa atgataagtt aatacgtgag
     5101 gttaaagtca ttactttaaa atcaaaactt gtctctgatt tcagaaagga tttccaattc
     5161 tataaagtta gagaaattaa taattatcat catgctcatg atgcatattt gaatgctgta
     5221 gttggaactg ctttaatcaa gaaataccct aaattagaat ctgaatttgt atatggtgat
     5281 tacaaagtct atgatgttag aaagatgatt gctaaatcag aacaagaaat tggtaaagct
     5341 acagctaaat acttctttta ctctaacatt atgaatttct ttaaaacaga aattactttg
     5401 gcaaacggtg aaattagaaa aagacctctt attgaaacaa atggtgagac tggagagata
     5461 gtttgggaca aagggcgtga tttcgctact gttagaaaag ttttatcaat gccacaagtt
     5521 aacattgtaa agaaaacaga ggttcaaact ggtggtttct caaaagaaag tattttgcct
     5581 aaaagaaata gtgataaatt gattgccaga aaaaaggatt gggatccaaa gaaatatggt
     5641 ggtttcgact caccaaccgt agcctattct gttttggttg tggcaaaggt tgaaaagggt
     5701 aaaagtaaaa agcttaaatc agtaaaagaa cttttgggta ttacaataat ggaaagaagt
     5761 tcctttgaaa agaaccctat tgattttttg gaagctaaag gttataagga agtaaagaag
     5821 gacttaataa tcaaattgcc taaatattct ttatttgaat tagaaaatgg gagaaaaaga
     5881 atgttggctt ctgctggaga attgcaaaag ggtaatgaat tagcattgcc ttccaaatat
     5941 gttaacttct tgtatttagc ttcacactat gaaaagttga aagggtcacc agaagataac
     6001 gagcaaaaac aattatttgt tgaacaacac aaacactact tagatgagat tatagaacaa
     6061 attagtgaat tcagtaaaag agtgatatta gctgatgcaa atttagataa agttttgtca
     6121 gcctataaca aacatagaga taagccaatt agagaacaag cagaaaacat tattcactta
     6181 tttaccctta ccaatttagg agcacctgct gctttcaagt attttgatac aacaattgat
     6241 cgtaaaagat atacctcaac aaaagaagtc ttagacgcca ccttaattca tcaatcaatc
     6301 actggattgt atgagacaag aattgatttg tctcaattgg gtggtgatga aggggctgat
     6361 cctaagaaga aaagaaaagt tgatccaaag aaaaagcgta aggtggatcc taagaaaaag
     6421 agaaaggttg actacaaaga ccatgacggt gattataaag atcatgacat cgactacaag
     6481 gatgacgatg acaagtgata atgactgcag agatccatcg acctgccgcc aagctaattc
     6541 cgggcgaatt tctgtcgagt catgtaatta gttatgtcac gcttacattc acgccctccc
     6601 cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt
     6661 tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt
     6721 tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt
     6781 ttgggacgct cgaaggcttt aatttgcggc cgggcccccc ctcgaggaag ttcctatact
     6841 ttctagagaa taggaacttc ggatccacta gttctagatt tttgcaagca tttaaatatt
     6901 gccaagtaaa aacttcaaat tttctttccc cttggaactt tgactttatt tttttgacag
     6961 attattttga cacacacaca ccaaatgtgt taccccttaa aacaaaaaaa cactttttta
     7021 caatttcttg gtatccagaa tcattctaag catcattcaa ttataatttc aatccaaaaa
     7081 agtagtttta gtttgacttg aaacgtcaac aaacacaaat ttcaaatcat aacctctcct
     7141 gttgcctgtc aacaacacac cataaggaga aggaatagga ggaggaggag atagaaactt
     7201 gcacggcacc acaaaacaca aaattgattt caaccaatac ggtgacaaca acaatagatt
     7261 tccgatagaa ataatgatta tcggaataag ctagctttgc tttgctttgc tttgcttttt
     7321 gacttgctct aatttttcga aaataataat ggagaaaagt tcaaggtgtt taatgcatca
     7381 actaaaacag aaaataatac attagactaa acttttaatc tttctagtac caataattca
     7441 cgcgtgcgtt ttaatcccaa tcatgaaatg aagaagttat ttcccttttt ctttcatcaa
     7501 aaaagaacta aattattttt taaattttag taaacaaaac ctggaaatcg gggaaaccgg
     7561 gggagggggg cagaaggtga aacgggtaat attgataaat ttaatctata attgataaag
     7621 ttaaatttaa attgatttga attgatttga attgaatgaa atgcatttga ataaacggca
     7681 tcaaactaaa aaaatataga tcacattcat agtaaaacga taacaaagaa caccacaatt
     7741 tatagcaatg ataataaaca tctaaaaaga aaagggtacg agaaggagaa tgaaaaaaaa
     7801 caataagcta gttcttaatc tgttcagata tctaatttca aaaaaaagaa tagtataaaa
     7861 ggatagttga ttcctcttgg ttgttgaaaa tttgaatgat atcaatcaat taatcaatca
     7921 aataacaaca acccactaga catcaccatt gtcgacatgc cacaatttga tatattatgt
     7981 aaaacaccac ctaaggtgct tgttcgtcag tttgtggaaa ggtttgaaag accttcaggt
     8041 gagaaaatag cattatgtgc tgctgaacta acctatttat gttggatgat tacacataac
     8101 ggaacagcaa tcaagagagc cacattcatg agctataata ctatcataag caattcgttg
     8161 agtttcgata ttgtcaataa atcactccag tttaaataca agacgcaaaa agcaacaatt
     8221 ttggaagcct cattaaagaa attgattcct gcttgggaat ttacaattat tccttactat
     8281 ggacaaaaac atcaatctga tatcactgat attgtaagta gtttgcaatt acagttcgaa
     8341 tcatcggaag aagcagataa ggggaatagc cacagtaaaa aaatgcttaa agcacttcta
     8401 agtgagggtg aaagcatctg ggagatcact gagaaaatac taaattcgtt tgagtatact
     8461 tcgagattta caaaaacaaa aactttatac caattcctct tcctagctac tttcatcaac
     8521 tgtggaagat tcagcgatat taagaacgtt gatccgaaat catttaaatt agtccaaaat
     8581 aagtatttgg gagtaataat ccagtgttta gtgacagaga caaagacaag cgttagtagg
     8641 cacatatact tctttagcgc aaggggtagg atcgatccac ttgtatattt ggatgaattt
     8701 ttgaggaatt ctgaaccagt cctaaaacga gtaaatagga ccggcaattc ttcaagcaat
     8761 aaacaggaat accaattatt aaaagataac ttagtcagat cgtacaataa agctttgaag
     8821 aaaaatgcgc cttattcaat ctttgctata aaaaatggcc caaaatctca cattggaaga
     8881 catttgatga cctcatttct ttcaatgaag ggcctaacgg agttgactaa tgttgtggga
     8941 aattggagcg ataagcgtgc ttctgccgtg gccaggacaa cgtatactca tcagataaca
     9001 gcaatacctg atcactactt cgcactagtt tctcggtact atgcatatga tccaatatca
     9061 aaggaaatga tagcattgaa ggatgagact aatccaattg aggagtggca gcatatagaa
     9121 cagctaaagg gtagtgctga aggaagcata cgataccccg catggaatgg gataatatca
     9181 caggaggtac tagactacct ttcatcctac ataaatagac gcatataaga gtgaaattct
     9241 ggaaatctgg aaatctggtt ttgtattctt gttattcttc tttttgttat tacatatata
     9301 acttgttact tttttaaaaa aatctttgta tattttataa atatataaaa ctaaatttaa
     9361 gaaaaagaga aaaatgtttt atttgagaga ttgaaatttt acttgaattt agcttagctt
     9421 ttataaagta ttattatgta aaaaaacaaa acaaatatac atcaaaaagt taagactata
     9481 aaatagccac ccaaggcatt tctatatctt gttgttgttg ttttcatctt ctgtatcaga
     9541 ggaacttatt ttattatttt cgtcacgggt attttctctt gtttgatgat tcatcccatt
     9601 cattccatca taaaatgtcg acactggatg gcggcgttag tatcgaatcg acagcagtat
     9661 agcgaccagc attcacatac gattgacgca tgatattact ttctgcgcac ttaacttcgc
     9721 atctgggcag atgatgtcga ggcgaaaaaa aatataaatc acgctaacat ttgattaaaa
     9781 tagaacaact acaatataaa aaaactatac aaatgacaag ttcttgaaaa caagaatctt
     9841 tttattgtca gtactgactc gagttattat ggacatggca tagacatata caaagcttgt
     9901 tcaccatcgg aagcagtacc atcgtataaa gcagtatcca aaccacacaa agtgaaaccc
     9961 attcttctat aagcatgaat agctggagca ttaacattgg taacttccaa ccacaaatga
    10021 ccagcacctc tttctctggc gaattcagta gccaaaccca tcaaagctct accaacacca
    10081 tgacctctat gttctggagc aacttcaata tcttcaacag tcaatcttct gttccaacca
    10141 gaataagaaa caacaacgaa accagccaaa tcaccatcat caccataagc aacgaaagtt
    10201 ctagaatctg gatcaccatc ttcaccagca tcggattcat catcggattc atcatctggg
    10261 aaaaccttag tcaatggtgg atcaactgga acttctctca aagtgaaacc atcaccagta
    10321 gcagtaactc taaaaacagt atcggtagtg aaagaaccat ccaaagcttc aatagcttca
    10381 gcatcacctg gaacagaagt tctgtatcta taagcagtat catccaaagt agtagacata
    10441 attgtaggat ccggttgttt atgttcggat gtgatgtgag aactgtatcc tagcaagatt
    10501 ttaaaaggaa gtatatgaaa gaagaacctc agtggcaaat cctaaccttt tatatttctc
    10561 tacaggggcg cggcgtgggg acaattcaac gcgtctgtga ggggagcgtt tccctgctcg
    10621 caggtctgca gcgaggagcc gtaatttttg cttcgcgccg tgcggccatc aaaatgtatg
    10681 gatgcaaatg attatacatg gggatgtatg ggctaaatgt acgggcgaca gtcacatcat
    10741 gcccctgagc tgcgcacgtc aagactgtca aggagggtat tctgggcctc catgtcgctg
    10801 gccgggtgac ccggcgggga cgaggcaagc ttgatgtgcg gccgcagtga ttagacttag
    10861 tccgttcaaa tcaagcacaa ctctgttcat tgtttcaaca agaattaatt caaaaacagg
    10921 ttcggtgcat aatttgcaaa aaaatattgc agcttctgtg gctcgaacac agtacctcca
    10981 gatttcaggt ttgaaatact tcagtctgac gctctcccag atgagctaaa gctgcaataa
    11041 gaaaacccac gccgggattc gaacccggaa tcctttgatt agaagtcaaa agcgataacc
    11101 atttcgccac gcaggcctac ttgatgggtt tgtaaatggt ctactttttc agacctaaca
    11161 gaaattttaa tgaaagtcat attcttatac aataaaactg tgtcataaaa gcagatattc
    11221 gactttcgta gattatatag gacccaagaa ctaaaattta atgccatatt atgcattttt
    11281 aatctgtaaa agtgttgttt ccaacctatc acaagtacgt tcttgtaact tgtgtttgta
    11341 gggttgcaaa tgaatcataa caacatctca acagaacatg tatagcaaag cttagtataa
    11401 aatcagtgtt ttgagaggca atccaagaat gtttacatca aagtttcaat aaatatcgac
    11461 cgaaactgaa aatcttttta ggttattgtt cacttttttg taaatattta aacatttttt
    11521 ggacctaaaa aaatacaagc accaattacg taccaagaag catctaatca actcccagat
    11581 caccactata catttaaaag tcattggtca ataactatac tcgagtattg cctcatcaaa
    11641 gaaacaatca aatattatag atactcactc catcacgtga taatttcact ggtatggaaa
    11701 agtggaaaat tttataaaaa aaaatttgat gcctttggca tagctgaaac ttcggcccaa
    11761 taggattgga gaatatgttt tcgcagcgtt cttacaatta aattgtggtg gaagttcgag
    11821 acttgcgtaa actattttta atttggagac ggaattccgt ctcgttttag agctagaaat
    11881 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
    11941 ttttttctcg agttttttta tcgagtgttt aaggataatg ataactgaag agaagaatta
    12001 gttttgccgc caccgcgtga agttcctatt ctctagaaag tataggaact tcccgcggga
    12061 agttcctata ctttctagag aataggaact tcacgcgggt ttgcctctga ttaaataaaa
    12121 aaaagctggt gctttttttt cttttatagg aacatcttga atatatgaac taattaaatg
    12181 ataatttttt acccatcttt actcttaatc actgagctgc agtcaaagaa aaagggatac
    12241 agcacctggt gaagagatga acggagacta acttagacgc gttgattctt ttcaattgca
    12301 cattttatta atcgatgcta acgtctattt acatatattc tttagagata ttatctaggg
    12361 cttcaaataa tctctggaca gcaataaaag tctcttcaaa agtattgtat aacggcaatg
    12421 gggctaatct gattacatct ggtcttcttt cgtcacagat tatagcatga tcatgcaagt
    12481 acgcattaac tcgttccatg acgttcttgc ccttttcatc gaaatgcggt tgaaacataa
    12541 tggacaattg acatcctctt tcagctggat tcaaaggagt taaaatttta aacccaaatt
    12601 tggagtttga tgtactggat tgtggtatgt aatacttgga attcgtcaat agatcctgta
    12661 aaaattgagt caaagcaaca cttttttcac gaagtttaga tactccaccc actttagcat
    12721 acacttccaa tgacgacttc acagcaacaa catcaagaac agaaggattt gactgtctgt
    12781 aagaaagagc cgagtttatt ggatcaaact cttctaacat tttgaatcgt tcttgggagt
    12841 tattgcccca ccaaccagct agtctaggaa cgaaactgct cttcttgttc tctatagtgt
    12901 atttttcatg cacaaaaatt ccacctatgg ctccaggtcc cgagtttaaa tatttgtagg
    12961 aacaccaagc agcaaaatct actccccaat catgtaaatt taatggaaca ttcccaactg
    13021 catgggcaag atcccaccca actttaattt gttggctctt ttccttagcg tatttagtta
    13081 tttcctctat cttgaaaaat tgaccagtgt agtattggat accaggaaaa cacactagag
    13141 ccaattcatc caggttctca tctatagcct tgattattct ttctgtttta atataagttt
    13201 caccaggttg aacttccaat tgaatcaaat gtttctcgtc gtatccgaac aatttaacaa
    13261 tgttcaaaaa tgcatagtag tcagaaggaa atgcttgttt ttcaaataaa attttggttc
    13321 ttttcccctc aggtttgtaa aaatggatca acaatgcatt caagtttgct gttaaagaac
    13381 ccataactgc aacttcgttt tcctttgcac caacaatggg ggctattaat ggtaataagg
    13441 gtaaatcgat gtctacccac ggtgttaaca gtttgtcagg atgattgaaa tgagactcaa
    13501 cccctcgttc aacccatgca ttcaattcat cattgatagc tttctttgta ttcttaggca
    13561 tcaacccaag agagtttcca cataaataaa tagactcagt tgatgactca tatttattat
    13621 ttttgatacc taatgatcca aaagttggta tggcaaactc atttttaaaa gttgggaact
    13681 ttttgtccaa tttctttgcc tcggctaatg acatctgata ataaaatggg gttggagtag
    13741 ttggtggtat aaccggagag atagaattga agaaaaaaat cggaaacaac aaaaaaagtt
    13801 gatacccagt attatgtggg agataattgc gaatggtgga aaaaaaaaag acgcctttgg
    13861 gcctcaacaa caattctaac agcttgagag ctttacaatc gagaaactat gattcattcc
    13921 gttttaatat gtatgtactt agtaaactcg tgaattttat ttgtggtcta ctttagtact
    13981 aacataatca ttggatagtc aataatgatg gtcttccgag actaatgaaa ttctatacca
    14041 aagtcgatat tccaacacag aaattgctct tgcaacaagt gcacctgttg atatctacta
    14101 gatcagcaca agagtgttcg gagctcgcat gcaggcctca gcttttgttc cctttagtga
    14161 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
    14221 ccgctcacaa ttccacacaa catacgagcc gggagcataa agtgtaaagc ctggggtgcc
    14281 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
    14341 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
    14401 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
    14461 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
    14521 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
    14581 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
    14641 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
    14701 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
    14761 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
    14821 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
    14881 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
    14941 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
    15001 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
    15061 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
    15121 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
    15181 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
    15241 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
    15301 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
    15361 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
    15421 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
    15481 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
    15541 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
    15601 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
    15661 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
    15721 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
    15781 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
    15841 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
    15901 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
    15961 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
    16021 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
    16081 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
    16141 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
    16201 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
    16261 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
    16321 tttc
//
