pV1081

LOCUS       pV1081       14621 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Solo vector for expression of CaCas9 and sgRNA for CaADE2 
            mutagenesis - integrates at ENO1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14621)
  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 14621)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2015"; volume: "1(3)"; pages: "e1500248"
COMMENT     Sequence Label: pV1081
FEATURES             Location/Qualifiers
     source          1..14621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(26..454)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(136..157)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     348..367
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     581..603
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     595..612
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     596..612
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     622..641
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        622..640
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2667..6770
                     /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             2667..6770
                     /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             6783..6803
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6807..6827
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6831..6851
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6852..6917
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     terminator      6982..7229
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6998..7016)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     misc_RNA        10155..10230
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(12429..12449)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(12429..12447)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(12468..12484)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12468..12484)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(12481..12503)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    12492..12508
                     /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(12516..12522,12523..12540,12541..12546))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    12561..12582
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12699..12716)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12870..13458)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12950..12969)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(13629..14420,14421..14489))
                     /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     14252..14271
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(14490..14594)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
       61 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      121 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
      181 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
      241 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
      301 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
      361 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
      421 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
      481 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
      541 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
      601 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattgag gcctgcatgc
      661 ggtaccttta tcagcaagta gaaaacaacc aaagctcttg aaattgtgca atgaagattt
      721 catcaaacat aaaaccatta tgaacgcttg gaagttgtta caacgaagaa gaataaccca
      781 acaatctgaa aaattgtcta agcaatataa aagcattgtc aatgccatgg aagatttgaa
      841 gcaaacaagt cccgaattgt tcgaagctgc aaatgctaaa gaccctaaac gtttcactac
      901 cttcccaata gagatgagag tgcctaccga ttatccacct aacaagccat ggacttacaa
      961 ctttgtttct tcaaaaaccc atcattagac tgggttcaga tgtaaataga tattatatta
     1021 taaatgtaca taatcgaata gattgttatt atttgttcaa ctcgtcctaa tcctccaata
     1081 ctctcgcctt tctttttcta ctaggtgtgc cactactacc actgggcgtc tcgttcttct
     1141 cgatactatt agctttactt cctgcactag cagtggttgg atcaacagaa tcttcataat
     1201 catcaaaatc gtcttttgaa gaccccccgt ttgatgtatg gccctgtctt ttcatcaaac
     1261 tttttatata gttgactgaa ctgaggctaa atatgtgatc atcttcacta tagacaatct
     1321 ttctcttatt tgcaccaccg ccaccaccag tctttgagaa attctcaaaa ccttttacga
     1381 tattaccaag cgggctctct tcgaaataat ctatctcttt ttgatatatc gaatcctcta
     1441 gcgtggttag ctttctagtt agttcttgct tcttaagaat ttgctggatt agtttatttt
     1501 tcaattcaac gtatttctca gagtcatctt tagattttga tgaagatgtg cgttcattcg
     1561 ctatatcctt cttggtcgtg tcttttcgat cctccttggc tggcactgaa ctcgtctttt
     1621 ttggcgttgc tgttccagac agacttatct cattagattt ggaacttgtg ggtttaacat
     1681 catttgcatc tttagtagac atgattgtgt aataccgtga ttgtttgttt tgaaaggtct
     1741 gtcatatttc tatcaatttc aaaacaaaat gttcatcaga aaaaagccaa aaatgtctct
     1801 tctagcttct tagtggtgtc gcaaaataca caatgtcgct caacaatcca cattcccggc
     1861 gcatagctca aatcacatga ctacagctaa caattacaca aaaaaattct cttttcgatg
     1921 tagcaactat cttcaactaa aacattttct ccttcggccc atgattgtcc tccgggtcga
     1981 cagcaagccg ttacaattga gatggaaagc gacctacctt cactcgataa ggtgcttaat
     2041 tgtacttcat ataaatctgg cccggatcta aacaaatgag ttccattaag ccgtgggttc
     2101 tcaattaggg tttttgtttt tgatttagaa aaaagagatc aagatttgtt tacaggtgat
     2161 gccttttttt agaacttatg cgttgcaaaa gttgactaac gatttctata aggtgatcca
     2221 cactaattat acaaacgtac aaacagacat acttttcctg cgttcacctg atgttggcca
     2281 gatttctctc ttcattgcat agaacataac cacactaggg caacagaaaa aaaaaaaaaa
     2341 agtgcatcgg gaagttgtgt tccattcatt atatgtctac tactgcatat gagtagccca
     2401 cccaccacca ccatagtaag tttttgtgta tgcgcgccgt caggttattt catttctgaa
     2461 tttttcaacc accttactcc ctttattgtt gattgacaat tttgctcaca gtaagatctt
     2521 ttagactcca attaatataa aataagtctg attttccaat tcctgttttt tctttttttt
     2581 tctgtttcta tttctttcct tttctccctt ttttttaatt cttcattcaa tcatcaattg
     2641 ataattcagg aatattacaa cccgggatgg ataaaaagta tagtattggt ttagatattg
     2701 gtactaactc tgtgggttgg gcagttatca ctgacgaata taaagttcca tcaaagaaat
     2761 ttaaggtgtt aggtaacact gacagacact caataaaaaa gaatcttatc ggtgctcttt
     2821 tgttcgactc cggtgaaact gccgaggcta cacgtttaaa aagaacagca agaagaagat
     2881 atacccgtag aaaaaataga atatgttatt tacaagaaat cttttctaat gaaatggcta
     2941 aagttgatga ttcctttttc catagattgg aagagtcatt tttggttgaa gaagacaaaa
     3001 agcatgagag acatccaatc tttgggaata tagttgatga agtggcttac catgaaaaat
     3061 atcctaccat ttatcattta agaaagaaat tggtagattc aactgataaa gctgacctta
     3121 gattaatcta tttagcactt gcccatatga ttaaatttag aggtcatttt ttgattgaag
     3181 gtgatttgaa cccagataat tctgacgtgg ataaattatt tattcaatta gtccaaacct
     3241 acaaccaatt atttgaggaa aatccaatta atgctagtgg tgtcgatgcc aaagctatat
     3301 tatcagccag attatcaaaa tctagacgtt tggaaaattt gattgcccaa ttgccaggag
     3361 aaaaaaagaa tggattattt ggaaacttga tcgcattatc attgggtttg acaccaaatt
     3421 ttaaatctaa ttttgattta gctgaagatg ctaaattaca attatcaaaa gacacctatg
     3481 acgacgattt ggacaattta cttgctcaaa ttggtgatca atatgcagat ttgttcttag
     3541 ctgctaaaaa cttatctgat gctattttgt tgtctgatat tttgagagtg aacacagaaa
     3601 taaccaaagc tccattatca gcatctatga tcaaacgtta tgatgaacac catcaggatt
     3661 tgactttatt gaaagctttg gtgagacaac aattgccaga gaagtataaa gaaatctttt
     3721 tcgatcaatc taaaaacggg tatgcaggtt atattgatgg gggtgcctcc caagaggaat
     3781 tttacaaatt tataaaacct attttagaaa agatggatgg gactgaggaa cttttggtca
     3841 aattgaacag agaagatttg ttacgtaaac agagaacttt tgataatggt agtatacctc
     3901 accaaattca tttgggtgag ttgcatgcaa ttttaagaag acaagaagat ttttatccat
     3961 ttttaaaaga taatagagaa aaaatcgaga aaattttaac ctttagaatt ccatactatg
     4021 ttgggccttt ggctagaggt aattcaagat ttgcctggat gacacgtaaa tcagaagaaa
     4081 ctattacccc ttggaatttt gaagaggttg ttgataaagg agcatcagca cagagtttta
     4141 ttgaaagaat gaccaatttc gataaaaact taccaaatga aaaagtttta ccaaaacatt
     4201 ccttgttata cgaatatttt actgtttaca atgaacttac aaaggttaaa tatgttactg
     4261 aaggtatgcg taagccagcc tttttatctg gagaacagaa aaaggcaata gttgatttat
     4321 tgtttaaaac aaatagaaaa gttactgtta aacaattaaa agaagattac tttaagaaaa
     4381 ttgaatgttt tgattcagtt gaaatcagtg gtgttgaaga cagatttaat gctagtttag
     4441 gaacttacca tgatttactt aaaattatca aagataaaga tttcttggat aacgaagaaa
     4501 atgaagacat tttagaagac attgttttaa ccttaacttt attcgaagat agagagatga
     4561 ttgaagaacg tttgaagact tatgcacatt tgtttgacga taaagtgatg aaacagttga
     4621 aaagaagacg ttatactgga tggggtagat tgtctcgtaa attgatcaat ggaattagag
     4681 ataaacaaag tggtaaaact atcttggact ttttgaaatc tgacggattt gctaatagaa
     4741 atttcatgca attgatccac gacgatagtt tgacatttaa agaagacatc caaaaggccc
     4801 aagtgagtgg gcaaggtgat tcattacatg aacatattgc aaatttagcc ggatctcctg
     4861 ctattaagaa agggatatta caaactgtta aagttgtgga tgaattagtg aaagtaatgg
     4921 gaagacataa acctgaaaac attgtcattg agatggcaag agaaaatcaa actacacaaa
     4981 aaggacagaa aaatagtaga gaacgtatga aaagaataga agagggtatt aaagaattgg
     5041 gtagtcaaat attgaaagaa cacccagtgg aaaataccca gttgcaaaat gaaaaattat
     5101 atctttacta ccttcaaaat ggacgtgata tgtatgttga tcaggaatta gatataaata
     5161 gactttcaga ttatgatgta gatcatatag ttccacaatc tttcttgaaa gatgattcca
     5221 tagacaataa agtattaact agaagtgata aaaatagagg taaaagtgat aatgtcccaa
     5281 gtgaggaagt cgtcaaaaag atgaaaaatt actggcgtca acttttgaat gctaaattaa
     5341 ttactcaaag aaaatttgat aatttgacta aagcagaaag aggtgggctt tctgaattag
     5401 ataaagccgg gttcattaaa agacaattgg tcgaaactag acaaattact aaacatgttg
     5461 cccaaatttt agattcccgt atgaacacta agtatgacga aaatgataag ttaatacgtg
     5521 aggttaaagt cattacttta aaatcaaaac ttgtctctga tttcagaaag gatttccaat
     5581 tctataaagt tagagaaatt aataattatc atcatgctca tgatgcatat ttgaatgctg
     5641 tagttggaac tgctttaatc aagaaatacc ctaaattaga atctgaattt gtatatggtg
     5701 attacaaagt ctatgatgtt agaaagatga ttgctaaatc agaacaagaa attggtaaag
     5761 ctacagctaa atacttcttt tactctaaca ttatgaattt ctttaaaaca gaaattactt
     5821 tggcaaacgg tgaaattaga aaaagacctc ttattgaaac aaatggtgag actggagaga
     5881 tagtttggga caaagggcgt gatttcgcta ctgttagaaa agttttatca atgccacaag
     5941 ttaacattgt aaagaaaaca gaggttcaaa ctggtggttt ctcaaaagaa agtattttgc
     6001 ctaaaagaaa tagtgataaa ttgattgcca gaaaaaagga ttgggatcca aagaaatatg
     6061 gtggtttcga ctcaccaacc gtagcctatt ctgttttggt tgtggcaaag gttgaaaagg
     6121 gtaaaagtaa aaagcttaaa tcagtaaaag aacttttggg tattacaata atggaaagaa
     6181 gttcctttga aaagaaccct attgattttt tggaagctaa aggttataag gaagtaaaga
     6241 aggacttaat aatcaaattg cctaaatatt ctttatttga attagaaaat gggagaaaaa
     6301 gaatgttggc ttctgctgga gaattgcaaa agggtaatga attagcattg ccttccaaat
     6361 atgttaactt cttgtattta gcttcacact atgaaaagtt gaaagggtca ccagaagata
     6421 acgagcaaaa acaattattt gttgaacaac acaaacacta cttagatgag attatagaac
     6481 aaattagtga attcagtaaa agagtgatat tagctgatgc aaatttagat aaagttttgt
     6541 cagcctataa caaacataga gataagccaa ttagagaaca agcagaaaac attattcact
     6601 tatttaccct taccaattta ggagcacctg ctgctttcaa gtattttgat acaacaattg
     6661 atcgtaaaag atatacctca acaaaagaag tcttagacgc caccttaatt catcaatcaa
     6721 tcactggatt gtatgagaca agaattgatt tgtctcaatt gggtggtgat gaaggggctg
     6781 atcctaagaa gaaaagaaaa gttgatccaa agaaaaagcg taaggtggat cctaagaaaa
     6841 agagaaaggt tgactacaaa gaccatgacg gtgattataa agatcatgac atcgactaca
     6901 aggatgacga tgacaagtga taatgactgc agagatccat cgacctgccg ccaagctaat
     6961 tccgggcgaa tttctgtcga gtcatgtaat tagttatgtc acgcttacat tcacgccctc
     7021 cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta
     7081 tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt
     7141 tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg
     7201 ttttgggacg ctcgaaggct ttaatttgcg gccgggcccc ccctaactca agtacaacag
     7261 atctggacca cctttgattg taaatagtaa taattaccac ccttatctaa ttatttattt
     7321 aacttattta tttatttatt atacatatat acaaatctaa taaagtgaaa atctccccct
     7381 tcacacttca catatgttag gcgtcatcct gtgctcccga gaaccagtac cagtacatcg
     7441 ctgtttcgtt cgagacttga ggtctagttt tatacgtgaa gaggtcaatg ccgccgagag
     7501 taaagccaca ttttgcgtac aaattgcagg caggtacatt gttcgtttgt gtctctaatc
     7561 gtatgccaag gagctgtctg cttagtgccc actttttcgc aaattcgatg agactgtgcg
     7621 cgactccttt gcctcggtgc gtgtgcgaca caacaatgtg ttcgatagag gctagatcgt
     7681 tccatgttga gttgagttca atcttcccga caagctcttg gtcgatgaat gcgccatagc
     7741 aagcagagtc ttcatcagag tcatcatctg agatgtaatc cttccggtag gggctcacac
     7801 ttctggtaga tagttcaaag ccttggtcgg ataggtgcac atcgaacact tcacgaacaa
     7861 tgaaatggtt ctcagcatcc aatgtttccg ccacctgctc agggatcacc gaaattttca
     7921 tatgagaacc gttatcgata actaaagcag caacttcttc tataaaaatg ggttagtatg
     7981 acagtcattt aaataaggaa tttttcagtt ggcttggttt caattcaatg ttcgtttttt
     8041 tttttcttgc tgtgtttgtg tttgtgttgt ttatagttgt gtgcactgat cgtcgaaaaa
     8101 aaaaattcat agtgagccgg gaaatctgta tagcccagat aacaacacaa gtccaaacta
     8161 gaaactcgtc aaacaccaaa agcaatgttg aatcaattgc cttgcacaag tacacgtagg
     8221 aaaacataaa acattgcaat tttgaatatt gagccttttg tcgtaacatt gattgatagg
     8281 attactcacc gaatggtttt gaaaccactg ccgacagatc aatcaatcaa tcaaaaaacg
     8341 tgaactttga aaaaggggaa gaacagatac attgaagtta gccatttcca ctgatcgtca
     8401 caacatatct gataaattac tttcaaaatt ataagctgat gtgtgtgtat tattaatgtg
     8461 acagtaacat cccaaacgag aaatattatc tcgacaacaa aaaagtttga tctgaattga
     8521 aaatgaagtt ttcccaccct acccatttgt catattgaaa ccaatcaact gattaatcaa
     8581 tcaattagaa ttgaagctaa actaaaacat accaccgtcc attttgaatg attatatttt
     8641 tttaatatta atatcgagat aatgtttcta agaaagaaag aaaaccagga gtgaaaatta
     8701 gaaaaggaaa ggaaaggaaa aaaagaaaaa tctgaaaata tataaaaaaa aattgtttcg
     8761 ttggcaataa atcttggtga gaacagcgac cgaaagcaaa taagaacaaa atatgagtgt
     8821 attacgttga acaactaatt aacgtgtgtg tatggatctt tttttctttt ttctctttaa
     8881 tcgactataa acaacaaaca tttttgggca gtgcacacac tacttaatat acacagcata
     8941 aattacacga ttagaaacaa attagcttat taaaataacc taatcaaacc gaatatttta
     9001 tggtattatg agtaaactat ataatataaa tagcacacac ccacaacaac aacaaaggaa
     9061 aactaaaagg ttttttcttt ttgaaaagat cgttttcttt attattctct agttttgacg
     9121 gcggccgcag tgattagact tagtccgttc aaatcaagca caactctgtt cattgtttca
     9181 acaagaatta attcaaaaac aggttcggtg cataatttgc aaaaaaatat tgcagcttct
     9241 gtggctcgaa cacagtacct ccagatttca ggtttgaaat acttcagtct gacgctctcc
     9301 cagatgagct aaagctgcaa taagaaaacc cacgccggga ttcgaacccg gaatcctttg
     9361 attagaagtc aaaagcgata accatttcgc cacgcaggcc tacttgatgg gtttgtaaat
     9421 ggtctacttt ttcagaccta acagaaattt taatgaaagt catattctta tacaataaaa
     9481 ctgtgtcata aaagcagata ttcgactttc gtagattata taggacccaa gaactaaaat
     9541 ttaatgccat attatgcatt tttaatctgt aaaagtgttg tttccaacct atcacaagta
     9601 cgttcttgta acttgtgttt gtagggttgc aaatgaatca taacaacatc tcaacagaac
     9661 atgtatagca aagcttagta taaaatcagt gttttgagag gcaatccaag aatgtttaca
     9721 tcaaagtttc aataaatatc gaccgaaact gaaaatcttt ttaggttatt gttcactttt
     9781 ttgtaaatat ttaaacattt tttggaccta aaaaaataca aacaccaatt acgtaccaag
     9841 aagcatctaa tcaactccca gatcaccact atacatttaa aagtcattgg tcaataacta
     9901 tactcgagta ttgcctcatc aaagaaacaa tcaaatatta tagatactca ctccatcacg
     9961 tgataatttc actggtatgg aaaagtggaa aattttataa aaaaaaattt gatgcctttg
    10021 gcatagctga aacttcggcc caataggatt ggagaatatg ttttcgcagc gttcttacaa
    10081 ttaaattgtg gtggaagttc gagacttgcg taaactattt ttaatttgga tccgcaacaa
    10141 tcatacgacc taatgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
    10201 caacttgaaa aagtggcacc gagtcggtgc tttttttctc gagttttttt atcgagtgtt
    10261 taaggataat gataactgaa gagaagaatt agttttgccg ccaccgcggc gaagaagaat
    10321 taggttctga agctatctac gctggtaaag atttccaaaa ggcttctcaa ttgtaagttt
    10381 gcctctgatt aaataaaaaa aagctggtgc ttttttttct tttataggaa catcttgaat
    10441 atatgaacta attaaatgat aattttttac ccatctttac tcttaatcac tgagctgcag
    10501 tcaaagaaaa agggatacag cacctggtga agagatgaac ggagactaac ttagacgcgt
    10561 tgattctttt caattgcaca ttttattaat cgatgctaac gtctatttac atatattctt
    10621 tagagatatt atctagggct tcaaataatc tctggacagc aataaaagtc tcttcaaaag
    10681 tattgtataa cggcaatggg gctaatctga ttacatctgg tcttctttcg tcacagatta
    10741 tagcatgatc atgcaagtac gcattaactc gttccatgac gttcttgccc ttttcatcga
    10801 aatgcggttg aaacataatg gacaattgac atcctctttc agctggattc aaaggagtta
    10861 aaattttaaa cccaaatttg gagtttgatg tactggattg tggtatgtaa tacttggaat
    10921 tcgtcaatag atcctgtaaa aattgagtca aagcaacact tttttcacga agtttagata
    10981 ctccacccac tttagcatac acttccaatg acgacttcac agcaacaaca tcaagaacag
    11041 aaggatttga ctgtctgtaa gaaagagccg agtttattgg atcaaactct tctaacattt
    11101 tgaatcgttc ttgggagtta ttgccccacc aaccagctag tctaggaacg aaactgctct
    11161 tcttgttctc tatagtgtat ttttcatgca caaaaattcc acctatggct ccaggtcccg
    11221 agtttaaata tttgtaggaa caccaagcag caaaatctac tccccaatca tgtaaattta
    11281 atggaacatt cccaactgca tgggcaagat cccacccaac tttaatttgt tggctctttt
    11341 ccttagcgta tttagttatt tcctctatct tgaaaaattg accagtgtag tattggatac
    11401 caggaaaaca cactagagcc aattcatcca ggttctcatc tatagccttg attattcttt
    11461 ctgttttaat ataagtttca ccaggttgaa cttccaattg aatcaaatgt ttctcgtcgt
    11521 atccgaacaa tttaacaatg ttcaaaaatg catagtagtc agaaggaaat gcttgttttt
    11581 caaataaaat tttggttctt ttcccctcag gtttgtaaaa atggatcaac aatgcattca
    11641 agtttgctgt taaagaaccc ataactgcaa cttcgttttc ctttgcacca acaatggggg
    11701 ctattaatgg taataagggt aaatcgatgt ctacccacgg tgttaacagt ttgtcaggat
    11761 gattgaaatg agactcaacc cctcgttcaa cccatgcatt caattcatca ttgatagctt
    11821 tctttgtatt cttaggcatc aacccaagag agtttccaca taaataaata gactcagttg
    11881 atgactcata tttattattt ttgataccta atgatccaaa agttggtatg gcaaactcat
    11941 ttttaaaagt tgggaacttt ttgtccaatt tctttgcctc ggctaatgac atctgataat
    12001 aaaatggggt tggagtagtt ggtggtataa ccggagagat agaattgaag aaaaaaatcg
    12061 gaaacaacaa aaaaagttga tacccagtat tatgtgggag ataattgcga atggtggaaa
    12121 aaaaaaagac gcctttgggc ctcaacaaca attctaacag cttgagagct ttacaatcga
    12181 gaaactatga ttcattccgt tttaatatgt atgtacttag taaactcgtg aattttattt
    12241 gtggtctact ttagtactaa cataatcatt ggatagtcaa taatgatggt cttccgagac
    12301 taatgaaatt ctataccaaa gtcgatattc caacacagaa attgctcttg caacaagtgc
    12361 acctgttgat atctactaga tcagcacaag agtgttcgga gctcgcatgc aggcctcagc
    12421 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
    12481 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag
    12541 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
    12601 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
    12661 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
    12721 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
    12781 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
    12841 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
    12901 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
    12961 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
    13021 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
    13081 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
    13141 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
    13201 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
    13261 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
    13321 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
    13381 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
    13441 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
    13501 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
    13561 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
    13621 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
    13681 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    13741 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    13801 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
    13861 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
    13921 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
    13981 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
    14041 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
    14101 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    14161 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    14221 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    14281 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    14341 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    14401 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    14461 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    14521 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    14581 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta a
//