LOCUS       sequence-323600-       12124 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Trifunctional Cas9-VPR fusion protein plasmid for simultaneous 
            transcriptional activation, transcriptional repression, and genome 
            editing in yeast .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12124)
  AUTHORS   Dong C, Jiang L, Xu S, Huang L, Cai J, Lian J, Xu Z
  TITLE     A Single Cas9-VPR Nuclease for Simultaneous Gene Activation, 
            Repression, and Editing in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2020 Sep 18;9(9):2252-2257. doi: 
            10.1021/acssynbio.0c00218. Epub 2020 Aug 28.
  PUBMED    32841560
REFERENCE   2  (bases 1 to 12124)
  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"; doi: 
            "10.1021/acssynbio.0c00218"; journalName: "ACS Synth Biol"; date: 
            "2020-09-18- 18"; volume: "9"; issue: "9"; pages: "2252-2257"
COMMENT     Sequence Label: pRS415-Cas9-VPR
FEATURES             Location/Qualifiers
     source          1..12124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..38)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     138..157
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(651..1745)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1746..2155)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(2593..2614)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2684..3083
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             3095..3118
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3122..3142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3143..7243
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTYKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSITHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7268..7288
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7322..7471
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             7496..7516
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7946..8302
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             8321..8890
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             8900..8926
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(9376..9392)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9376..9392)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9389..9411)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9400..9416
                     /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(9424..9430,9431..9448,9449..9454))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9469..9490
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9607..9624)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9778..10366)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9858..9877)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10537..11328,11329..11397))
                     /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     11160..11179
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11398..11502)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    11539..12042
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     12084..12102
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
       61 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
      121 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatcgacta
      181 cgtcgttaag gccgtttctg acagagtaaa attcttgagg gaactttcac cattatggga
      241 aatggttcaa gaaggtattg acttaaactc catcaaatgg tcaggtcatt gagtgttttt
      301 tatttgttgt attttttttt ttttagagaa aatcctccaa tatataaatt aggaatcata
      361 gtttcatgat tttctgttac acctaacttt ttgtgtggtg ccctcctcct tgtcaatatt
      421 aatgttaaag tgcaattctt tttccttatc acgttgagcc attagtatca atttgcttac
      481 ctgtattcct ttacatcctc ctttttctcc ttcttgataa atgtatgtag attgcgtata
      541 tagtttcgtc taccctatga acatattcca ttttgtaatt tcgtgtcgtt tctattatga
      601 atttcattta taaagtttat gtacaaatat cataaaaaaa gagaatcttt ttaagcaagg
      661 attttcttaa cttcttcggc gacagcatca ccgacttcgg tggtactgtt ggaaccacct
      721 aaatcaccag ttctgatacc tgcatccaaa acctttttaa ctgcatcttc aatggcctta
      781 ccttcttcag gcaagttcaa tgacaatttc aacatcattg cagcagacaa gatagtggcg
      841 atagggttga ccttattctt tggcaaatct ggagcagaac cgtggcatgg ttcgtacaaa
      901 ccaaatgcgg tgttcttgtc tggcaaagag gccaaggacg cagatggcaa caaacccaag
      961 gaacctggga taacggaggc ttcatcggag atgatatcac caaacatgtt gctggtgatt
     1021 ataataccat ttaggtgggt tgggttctta actaggatca tggcggcaga atcaatcaat
     1081 tgatgttgaa ccttcaatgt agggaattcg ttcttgatgg tttcctccac agtttttctc
     1141 cataatcttg aagaggccaa aacattagct ttatccaagg accaaatagg caatggtggc
     1201 tcatgttgta gggccatgaa agcggccatt cttgtgattc tttgcacttc tggaacggtg
     1261 tattgttcac tatcccaagc gacaccatca ccatcgtctt cctttctctt accaaagtaa
     1321 atacctccca ctaattctct gacaacaacg aagtcagtac ctttagcaaa ttgtggcttg
     1381 attggagata agtctaaaag agagtcggat gcaaagttac atggtcttaa gttggcgtac
     1441 aattgaagtt ctttacggat ttttagtaaa ccttgttcag gtctaacact accggtaccc
     1501 catttaggac cacccacagc acctaacaaa acggcatcag ccttcttgga ggcttccagc
     1561 gcctcatctg gaagtggaac acctgtagca tcgatagcag caccaccaat taaatgattt
     1621 tcgaaatcga acttgacatt ggaacgaaca tcagaaatag ctttaagaac cttaatggct
     1681 tcggctgtga tttcttgacc aacgtggtca cctggcaaaa cgacgatctt cttaggggca
     1741 gacattagaa tggtatatcc ttgaaatata tatatatata tattgctgaa atgtaaaagg
     1801 taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac aataggtcct
     1861 taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga acgcttctct
     1921 attctatatg aaaagccggt tccggcgctc tcacctttcc tttttctccc aatttttcag
     1981 ttgaaaaagg tatatgcgtc aggcgacctc tgaaattaac aaaaaatttc cagtcatcga
     2041 atttgattct gtgcgatagc gcccctgtgt gttctcgtta tgttgaggaa aaaaataatg
     2101 gttgctaaga gattcgaact cttgcatctt acgatacctg agtattccca cagttaactg
     2161 cggtcaagat atttcttgaa tcaggcgcct tagaccgctc ggccaaacaa ccaattactt
     2221 gttgagaaat agagtataat tatcctataa atataacgtt tttgaacaca catgaacaag
     2281 gaagtacagg acaattgatt ttgaagagaa tgtggatttt gatgtaattg ttgggattcc
     2341 atttttaata aggcaataat attaggtatg tagatatact agaagttctc ctcgaccggt
     2401 cgatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt
     2461 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2521 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2581 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2641 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgcatagct tcaaaatgtt
     2701 tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca
     2761 aaacacccaa gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt
     2821 acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga
     2881 aaaaggcaat aaaaattttt atcacgtttc tttttcttga aaattttttt ttttgatttt
     2941 tttctctttc gatgacctcc cattgatatt taagttaata aacggtcttc aatttctcaa
     3001 gtttcagttt catttttctt gttctattac aacttttttt acttcttgct cattagaaag
     3061 aaagcatagc aatctaatct aagttttcta gatggattat aaagatgacg atgacaaacc
     3121 tccaaaaaag aagagaaagg tcgataagaa atactcaata ggcttagata tcggcacaaa
     3181 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt
     3241 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga
     3301 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg
     3361 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga
     3421 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga
     3481 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac
     3541 tatctatcat ctgcgaaaaa aattggtaga ttctacttat aaagcggatt tgcgcttaat
     3601 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt
     3661 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca
     3721 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc
     3781 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa
     3841 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc
     3901 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga
     3961 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa
     4021 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa
     4081 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct
     4141 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca
     4201 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa
     4261 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa
     4321 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctatta cccatcaaat
     4381 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa
     4441 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc
     4501 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac
     4561 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg
     4621 catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct
     4681 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat
     4741 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa
     4801 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg
     4861 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta
     4921 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga
     4981 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga
     5041 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg
     5101 ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca
     5161 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat
     5221 gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc
     5281 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa
     5341 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca
     5401 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca
     5461 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca
     5521 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta
     5581 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag
     5641 tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa
     5701 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga
     5761 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca
     5821 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc
     5881 tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat
     5941 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa
     6001 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa
     6061 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg
     6121 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa
     6181 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc
     6241 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa
     6301 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg
     6361 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat
     6421 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag
     6481 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt
     6541 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc
     6601 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt
     6661 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt
     6721 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct
     6781 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa
     6841 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca
     6901 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag
     6961 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata
     7021 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac
     7081 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa
     7141 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg
     7201 tctttatgaa acacgcattg atttgagtca gctaggaggt gacccaattg ccggatccaa
     7261 ggctagcccg aaaaagaaac gcaaagttgg gcgcgccgag gccagcggtt ccggacgggc
     7321 tgacgcattg gacgattttg atctggatat gctgggaagt gacgccctcg atgattttga
     7381 ccttgacatg cttggttcgg atgcccttga tgactttgac ctcgacatgc tcggcagtga
     7441 cgcccttgat gatttcgacc tggacatgct gattaactct agaagttccg gatctccgaa
     7501 aaagaaacgc aaagttggta gccagtacct gcccgacacc gacgaccggc accggatcga
     7561 ggaaaagcgg aagcggacct acgagacatt caagagcatc atgaagaagt cccccttcag
     7621 cggccccacc gaccctagac ctccacctag aagaatcgcc gtgcccagca gatccagcgc
     7681 cagcgtgcca aaacctgccc cccagcctta ccccttcacc agcagcctga gcaccatcaa
     7741 ctacgacgag ttccctacca tggtgttccc cagcggccag atctctcagg cctctgctct
     7801 ggctccagcc cctcctcagg tgctgcctca ggctcctgct cctgcaccag ctccagccat
     7861 ggtgtctgca ctggctcagg caccagcacc cgtgcctgtg ctggctcctg gacctccaca
     7921 ggctgtggct ccaccagccc ctaaacctac acaggccggc gagggcacac tgtctgaagc
     7981 tctgctgcag ctgcagttcg acgacgagga tctgggagcc ctgctgggaa acagcaccga
     8041 tcctgccgtg ttcaccgacc tggccagcgt ggacaacagc gagttccagc agctgctgaa
     8101 ccagggcatc cctgtggccc ctcacaccac cgagcccatg ctgatggaat accccgaggc
     8161 catcacccgg ctcgtgacag gcgctcagag gcctcctgat ccagctcctg cccctctggg
     8221 agcaccaggc ctgcctaatg gactgctgtc tggcgacgag gacttcagct ctatcgccga
     8281 tatggatttc tcagccttgc tgggctctgg cagcggcagc cgggattcca gggaagggat
     8341 gtttttgccg aagcctgagg ccggctccgc tattagtgac gtgtttgagg gccgcgaggt
     8401 gtgccagcca aaacgaatcc ggccatttca tcctccagga agtccatggg ccaaccgccc
     8461 actccccgcc agcctcgcac caacaccaac cggtccagta catgagccag tcgggtcact
     8521 gaccccggca ccagtccctc agccactgga tccagcgccc gcagtgactc ccgaggccag
     8581 tcacctgttg gaggatcccg atgaagagac gagccaggct gtcaaagccc ttcgggagat
     8641 ggccgatact gtgattcccc agaaggaaga ggctgcaatc tgtggccaaa tggacctttc
     8701 ccatccgccc ccaaggggcc atctggatga gctgacaacc acacttgagt ccatgaccga
     8761 ggatctgaac ctggactcac ccctgacccc ggaattgaac gagattctgg ataccttcct
     8821 gaacgacgag tgcctcttgc atgccatgca tatcagcaca ggactgtcca tcttcgacac
     8881 atctctgttt ttaattaact acccgtacga cgttccggac tacgcttctt gagatccttt
     8941 gcgaacactt ttattaattc atgatcacgc tctaatttgt gcatttgaaa tgtactctaa
     9001 ttctaatttt atatttttaa tgatatcttg aaaagtaaat acgtttttaa tatatacaaa
     9061 ataatacagt ttaattttca agtttttgat catttgttct cagaaagttg agtgggacgg
     9121 agacaaagaa actttaaaga gaaatgcaaa gtgggaagaa gtcagttgtt taccgaccgc
     9181 actgttattc acaaatattc caattttgcc tgcagactca cgtccacaaa ttttggttag
     9241 tttggtaaat ggtaaggata tagtagagcc tttttgaaat gggaaatatc ttctttttct
     9301 gtatcccgct tcaaaaagtg tctaatgagt cagttatctt tagtgagggt taattgcgcg
     9361 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     9421 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     9481 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     9541 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     9601 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     9661 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     9721 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     9781 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     9841 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     9901 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     9961 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
    10021 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
    10081 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
    10141 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
    10201 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
    10261 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
    10321 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
    10381 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
    10441 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
    10501 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
    10561 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
    10621 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
    10681 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
    10741 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
    10801 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
    10861 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
    10921 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
    10981 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
    11041 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
    11101 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
    11161 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
    11221 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
    11281 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
    11341 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
    11401 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
    11461 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
    11521 gccacctggg tccttttcat cacgtgctat aaaaataatt ataatttaaa ttttttaata
    11581 taaatatata aattaaaaat agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt
    11641 tttccgaaga tgtaaaagac tctaggggga tcgccaacaa atactacctt ttatcttgct
    11701 cttcctgctc tcaggtatta atgccgaatt gtttcatctt gtctgtgtag aagaccacac
    11761 acgaaaatcc tgtgatttta cattttactt atcgttaatc gaatgtatat ctatttaatc
    11821 tgcttttctt gtctaataaa tatatatgta aagtacgctt tttgttgaaa ttttttaaac
    11881 ctttgtttat ttttttttct tcattccgta actcttctac cttctttatt tactttctaa
    11941 aatccaaata caaaacataa aaataaataa acacagagta aattcccaaa ttattccatc
    12001 attaaaagat acgaggcgcg tgtaagttac aggcaagcga tccgtcctaa gaaaccatta
    12061 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
    12121 tcgg
//
