pDuRCC-N

LOCUS       pDuRCC-N              10325 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expression of Cas9 and two gRNA cassettes in S. cerevisiae; 
            Nourseothricin Resistance.
ACCESSION   .
VERSION     .
KEYWORDS    pDuRCC-N
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10325)
  AUTHORS   Generoso WC, Gottardi M, Oreb M, Boles E
  TITLE     Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae.
  JOURNAL   J Microbiol Methods. 2016 Aug;127:203-5. doi: 
            10.1016/j.mimet.2016.06.020. Epub 2016 Jun 17.
  PUBMED    27327211
REFERENCE   2  (bases 1 to 10325)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10325
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(10..29)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        53..321
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        345..419
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      424..443
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(496..514)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      555..1897
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    887..934
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1924..2028
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2029..2889
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2247..2266)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3060..3648
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3549..3568
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3685..3953
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        3977..4051
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4056..4075
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      4096..4343
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     4309..4327
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(4359..4379)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4392..8495)
                     /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"
     gene            complement(9184..10303)
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     terminator      9184..9381
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(9387..9959)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     promoter        complement(9960..10303)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
ORIGIN
        1 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccgggta attctttgaa
       61 aagataatgt atgattatgc tttcactcat atttatacag aaacttgatg ttttctttcg
      121 agtatataca aggtgattac atgtacgttt gaagtacaac tctagatttt gtagtgccct
      181 cttgggctag cggtaaaggt gcgcattttt tcacacccta caatgttctg ttcaaaagat
      241 tttggtcaaa cgctgtagaa gtgaaagttg gtgcgcatgt ttcggcgttc gaaacttctc
      301 cgcagtgaaa gataaatgat cggatccacg ttctctgcgg ccgcgtttta gagctagaaa
      361 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgg
      421 tgcttttttt gttttttatg tctcggcgaa ttgggtgcgg ccgcgtttgt cacctttcat
      481 aggcctgacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
      541 tggtttctta gtatgatcca atatcaaagg aaatgatagc attgaaggat gagactaatc
      601 caattgagga gtggcagcat atagaacagc taaagggtag tgctgaagga agcatacgat
      661 accccgcatg gaatgggata atatcacagg aggtactaga ctacctttca tcctacataa
      721 atagacgcat ataagtacgc atttaagcat aaacacgcac tatgccgttc ttctcatgta
      781 tatatatata caggcaacac gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg
      841 cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc
      901 gaagttccta ttctctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc
      961 tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata
     1021 ttgcgaatac cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg
     1081 ctatatccct atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg
     1141 acctctacat tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa
     1201 gaactattca tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt
     1261 atatagagac aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac
     1321 gctatcactt tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg
     1381 atgcctttat cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag
     1441 tggagtcagg ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct
     1501 taacggacct acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga
     1561 aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag
     1621 aacaaaaaag aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt
     1681 tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt
     1741 tgttttacaa aaatgaagca cagattcttc gttggtaaaa tagcgctttc gcgttgcatt
     1801 tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc
     1861 atttttgttc tacaaaatga agcacagatg cttcgttcag gtggcacttt tcggggaaat
     1921 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1981 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2041 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2101 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     2161 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     2221 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     2281 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     2341 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     2401 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     2461 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     2521 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     2581 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     2641 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     2701 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     2761 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     2821 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     2881 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     2941 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3001 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3061 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3121 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3181 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3241 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3301 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3361 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3421 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3481 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3541 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3601 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3661 gcgggctagg gataacaggg taattctttg aaaagataat gtatgattat gctttcactc
     3721 atatttatac agaaacttga tgttttcttt cgagtatata caaggtgatt acatgtacgt
     3781 ttgaagtaca actctagatt ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt
     3841 tttcacaccc tacaatgttc tgttcaaaag attttggtca aacgctgtag aagtgaaagt
     3901 tggtgcgcat gtttcggcgt tcgaaacttc tccgcagtga aagataaatg atcggatcca
     3961 cgttctctgc ggccgcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     4021 atcaacttga aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtctcggcg
     4081 aattgggtac cggccgcaaa ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt
     4141 tttcagtata atgttacatg cgtacacgcg tctgtacaga aaaaaaagaa aaatttgaaa
     4201 tataaataac gttcttaata ctaacataac tataaaaaaa taaataggga cctagacttc
     4261 aggttgtcta actccttcct tttcggttag agcggatgtg gggggagggc gtgaatgtaa
     4321 gcgtgacata actaattaca tgactcgagg tcgacttaaa cctttctctt cttctttggg
     4381 tcagctctag agtcaccacc caattgagac aagtcaattc tggtttcgta caaaccggta
     4441 atagattggt gaatcaaggt agcgtccaaa acttccttgg tagaggtgta tctctttctg
     4501 tcaatggtgg tgtcgaagta cttgaaagca gctggagcac ccaagttggt caaggtgaac
     4561 aagtgaataa tgttttcagc ttgttctcta attggcttgt ctctgtgctt gttgtaagca
     4621 gacaaaacct tgtccaagtt agcgtcagcc aaaataactc tcttagagaa ttcagaaatt
     4681 tgttcaataa tttcgtccaa gtagtgcttg tgttgttcaa cgaacaattg cttttgttcg
     4741 ttgtcttctg gagaaccctt caacttttcg tagtgagaag ccaagtacaa gaagttaacg
     4801 tacttagatg gcaaagccaa ttcgttaccc ttttgcaatt caccagcaga agccaacatt
     4861 ctctttctac cgttttccaa ttcgaacaaa gagtactttg gcaacttaat aatcaagtcc
     4921 ttcttaactt ccttgtaacc cttagcttcc aagaagtcaa ttgggttctt ttcgaaagaa
     4981 gatctttcca taatggtaat acccaacaat tccttaacag acttcaactt cttagactta
     5041 cccttttcaa ccttagcaac aaccaaaaca gagtaagcaa cggttggaga gtcgaaacca
     5101 ccgtacttct ttgggtccca gtccttcttt ctagcaatca acttgtcaga gtttctcttt
     5161 ggcaaaatag attccttaga gaaaccaccg gtttgaactt cggtcttctt aacaatgtta
     5221 acttgtggca tagacaaaac ctttctaacg gtagcgaagt ctctaccctt gtcccaaaca
     5281 atttcaccgg tttcaccgtt ggtttcaatc aatggtctct ttctaatttc accgttagcc
     5341 aaggtaattt cggtcttgaa gaagttcata atgttagagt agaagaagta cttagcggta
     5401 gccttaccaa tttcttgttc agacttagca atcatctttc taacgtcgta aaccttgtag
     5461 tcaccgtaaa cgaattcaga ttccaacttt gggtacttct taatcaaagc ggtaccaaca
     5521 acagcgttca agtaagcgtc gtgagcgtgg tggtagttgt taatttctct aaccttgtag
     5581 aattggaagt cctttctgaa gtcagaaacc aacttagact tcaaggtaat aaccttaact
     5641 tctctaatca acttgtcgtt ttcgtcgtac ttggtgttca ttctagagtc caaaatttga
     5701 gcaacgtgct tggtaatttg tctggtttca accaattgtc tcttaatgaa accagccttg
     5761 tccaattcag acaaaccacc tctttcagcc ttggtcaagt tgtcgaactt tctttgggta
     5821 atcaacttag cgttcaacaa ttgtctccag tagttcttca tcttcttaac aacttcttca
     5881 gatggaacgt tgtcagactt acctctgttc ttgtcagatc tggtcaaaac cttgttgtca
     5941 atagagtcgt ccttcaagaa agattgtgga acaatgtggt caacgtcgta gtcagacaat
     6001 ctgttaatgt ccaattcttg gtcaacgtac atgtctctac cgttttgcaa gtagtacaag
     6061 tacaactttt cgttttgcaa ttgggtgttt tcaactgggt gttccttcaa aatttgagaa
     6121 cccaattcct taataccttc ttcaattctc ttcattcttt ctctagagtt cttttgaccc
     6181 ttttgggtgg tttggttttc tctagccatt tcaataacaa tgttttctgg cttgtgtcta
     6241 cccataacct taaccaattc gtcaacaacc ttaacggttt gcaaaatacc cttcttaata
     6301 gctggagaac cagccaagtt agcaatgtgt tcgtgcaaag agtcaccttg accagaaact
     6361 tgagcctttt gaatgtcttc cttgaaggtc aaagagtcgt cgtgaatcaa ttgcatgaag
     6421 tttctgttag cgaaaccgtc agacttcaag aagtccaaaa tggtcttacc agattgcttg
     6481 tctctaatac cgttaatcaa ctttctagac aatctacccc aaccggtgta tcttcttctc
     6541 ttcaattgct tcataacctt gtcgtcgaac aagtgagcgt aggtcttcaa tctttcttca
     6601 atcatttctc tgtcttcgaa caaggtcaag gtcaaaacaa tgtcttccaa aatgtcttcg
     6661 ttttcttcgt tgtccaagaa gtccttgtcc ttaataatct tcaacaagtc gtggtaggta
     6721 cccaaagaag cgttgaatct gtcttcaaca ccagaaattt caacagagtc gaaacattca
     6781 atcttcttga agtagtcttc cttcaattgc ttaacggtaa cctttctgtt ggtcttgaac
     6841 aacaagtcaa caatagcctt cttttgttca ccagacaaga aagctggctt tctcatacct
     6901 tcggtaacgt acttaacctt ggtcaattcg ttgtaaacgg tgaagtattc gtacaacaaa
     6961 gagtgctttg gcaaaacctt ttcgtttggc aagttcttgt cgaagttggt cattctttca
     7021 atgaaagatt gagcagaagc acccttgtca acaacttctt cgaagttcca tggggtaatg
     7081 gtttcttcag actttctggt catccaagcg aatctagagt tacctctagc caatggacca
     7141 acgtagtatg gaattctgaa ggtcaaaatc ttttcaatct tttctctgtt gtccttcaag
     7201 aatgggtaga agtcttcttg tcttctcaaa atagcgtgca attcacccaa gtgaatttgg
     7261 tgtggaatag aaccgttgtc gaaggttctt tgctttctca acaagtcttc tctgttcaac
     7321 ttaaccaaca attcttcggt accgtccatc ttttccaaaa ttggcttaat gaacttgtag
     7381 aattcttctt gagaagcacc accgtcaatg taaccagcgt aaccgttctt agattggtcg
     7441 aagaaaattt ccttgtactt ttctggcaat tgttgtctaa ccaaagcctt caacaaggtc
     7501 aagtcttggt ggtgttcgtc gtatctctta atcatagaag cagacaatgg agccttggta
     7561 atttcggtgt taactctcaa aatgtcagac aacaaaatag cgtcagacaa gttcttagca
     7621 gccaagaaca agtcagcgta ttggtcacca atttgagcca acaagttgtc caagtcgtcg
     7681 tcgtaggtgt ccttagacaa ttgcaactta gcgtcttcag ccaagtcgaa gttagacttg
     7741 aagtttgggg tcaaacccaa agacaaagca atcaagttac cgaacaaacc gttcttcttt
     7801 tcacctggca attgagcaat caagttttcc aatcttctag acttagacaa tctagcagac
     7861 aaaatagcct tagcgtcaac accagaagcg ttaattgggt tttcttcgaa caattggttg
     7921 taggtttgaa ccaattgaat gaacaacttg tcaacgtcag agttgtctgg gttcaagtca
     7981 ccttcaatca agaagtgacc tctgaactta atcatgtgag ccaaagccaa gtaaatcaat
     8041 ctcaagtcag ccttgtcggt agagtcaacc aacttctttc tcaagtggta aatggttggg
     8101 tacttttcgt ggtaagcaac ttcgtcaaca atgttaccga aaattgggtg tctttcgtgc
     8161 ttcttgtctt cttcaaccaa gaaagattct tccaatctgt ggaagaaaga gtcgtcaacc
     8221 ttagccattt cgttagagaa aatttcttgc aagtaacaaa ttctgttctt tcttctggtg
     8281 tatcttcttc tagcggttct cttcaatctg gtagcttcag cggtttcacc agagtcgaac
     8341 aacaaagcac caatcaagtt cttcttaata gagtgtctgt cggtgttacc caaaaccttg
     8401 aacttcttag atggaacctt gtattcgtcg gtaataacag cccaaccaac agagttggta
     8461 ccaatgtcca aaccaataga gtacttcttg tccatatgta ttatcgtatt cctttttcgt
     8521 tcacttacta ttgctattcg cactcactac agatctatat tttatgttcc accctgttgt
     8581 cagatcagga aaggaatgaa gaggctctat ttaaaccaaa tacacctctt cgtttctcaa
     8641 tattgttggg ctttcctttt ttttttcctt ccacgtctat ctattgtctc tatcaaaatt
     8701 aggaaaaaga aatgaagaac gatgagccaa acgaggctga gacaatcatg catccgtaca
     8761 ttctatgtat gtttgggtgt atctttccag attcggttct tttttttttt cagtccaatt
     8821 gaaaaaaaaa aggaacagga acaacgggtc gaaatttgcc gtcacattca gagcgaagag
     8881 cgacagggga attggtgggc taaccacctg ttgagcccag ctagcagctg tcggtaaacc
     8941 gtcgttgggc ggggcgcgac aatgcagcca gcggaggcat tgtatggttt ccgtctggag
     9001 agtgggctta gccggcctgg cactccttct ctaagcagag tgagaaggga atctttttac
     9061 tggagaagag tgtttgaatc cagcagaagg taatacgctg cggtattttc tccttacgca
     9121 tctgtgcggt atttcacacc gcgttttcga cactggatgg cggcgttagt atcgaatcga
     9181 cagcagtata gcgaccagca ttcacatacg attgacgcat gatattactt tctgcgcact
     9241 taacttcgca tctgggcaga tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt
     9301 tgattaaaat agaacaacta caatataaaa aaactataca aatgacaagt tcttgaaaac
     9361 aagaatcttt ttattgtcag tactgattag gggcagggca tgctcatgta gagcgcctgc
     9421 tcgccgtccg aggcggtgcc gtcgtacagg gcggtgtcca ggccgcagag ggtgaacccc
     9481 atccgccggt acgcgtggat cgccggtgcg ttgacgttgg tgacctccag ccagaggtgc
     9541 ccggcgcccc gctcgcgggc gaactccgtc gcgagcccca tcaacgcgcg cccgaccccg
     9601 tgcccccggt gctccggggc gacctcgatg tcctcgacgg tcagccggcg gttccagccg
     9661 gagtacgaga cgaccacgaa gcccgccagg tcgccgtcgt ccccgtacgc gacgaacgtc
     9721 cgggagtccg ggtcgccgtc ctccccgtcg tccgattcgt cgtccgattc gtcgtcgggg
     9781 aacaccttgg tcaggggcgg gtccaccggc acctcccgca gggtgaagcc gtccccggtg
     9841 gcggtgacgc ggaagacggt gtcggtggtg aaggacccat ccagtgcctc gatggcctcg
     9901 gcgtcccccg ggacactggt gcggtaccgg taagccgtgt cgtcaagagt ggtacccatg
     9961 gttgtttatg ttcggatgtg atgtgagaac tgtatcctag caagatttta aaaggaagta
    10021 tatgaaagaa gaacctcagt ggcaaatcct aaccttttat atttctctac aggggcgcgg
    10081 cgtggggaca attcaacgcg tctgtgaggg gagcgtttcc ctgctcgcag gtctgcagcg
    10141 aggagccgta atttttgctt cgcgccgtgc ggccatcaaa atgtatggat gcaaatgatt
    10201 atacatgggg atgtatgggc taaatgtacg ggcgacagtc acatcatgcc cctgagctgc
    10261 gcacgtcaag actgtcaagg agggtattct gggcctccat gtcgctgatt accctgttat
    10321 cccta
//