LOCUS       sequence-107786-       10815 bp    DNA     circular SYN 14-APR-2025
DEFINITION  encodes yeast-optimized dCas9 synthetic transcription factor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10815)
  AUTHORS   Farzadfard F, Perli SD, Lu TK
  TITLE     Tunable and Multifunctional Eukaryotic Transcription Factors Based 
            on CRISPR/Cas.
  JOURNAL   ACS Synth Biol. 2013 Sep 11.
  PUBMED    23977949
REFERENCE   2  (bases 1 to 10815)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2013 Sep 11."
COMMENT     Sequence Label: pTPGI_dCas9
FEATURES             Location/Qualifiers
     source          1..10815
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        187..467
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             468..1142
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1241..1696)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1378..1399)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1590..1609
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1779..1944)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1865..1889
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2088..2711)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     2619..2638
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        complement(2729..3135)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        3643..4084
                     /label=TPGI promoter
                     /note="S. cerevisiae GAL1 promoter modified to contain two 
                     copies of the tet operator (Ellis et al., 2009)"
     protein_bind    3643..3760
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     protein_bind    3968..3986
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3989..4007
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4055..4078
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             4124..4144
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(4154..4177,4178..4180,4181..6667,6668..6670,
                     6671..8257)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     terminator      8284..8531
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(8300..8318)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        complement(8555..8573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8594..8610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8594..8610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8607..8629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8618..8634
                     /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(8642..8648,8649..8666,8667..8672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(8825..8842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8996..9584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(9755..10546,10547..10615))
                     /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     10378..10397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10616..10720)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10788..10806
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat
      241 atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa
      301 aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa
      361 gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat
      421 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta
      481 atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg
      541 cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa
      601 agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa
      661 atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg
      721 ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt
      781 ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag
      841 actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg
      901 attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg
      961 gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa
     1021 atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg
     1081 agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat
     1141 aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga
     1201 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt
     1261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     1321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     1381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     1441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     1501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     1561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     1621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     1681 ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     1741 tcggtgcggg cctcttcgct attacgccag ctgaattgga gcgacctcat gctatacctg
     1801 agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc gttttaaaac
     1861 ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt taataataaa
     1921 aatcataaat cataagaaat tcgcttattt agaagtgtca acaacgtatc taccaacgat
     1981 ttgacccttt tccatctttt cgtaaatttc tggcaaggta gacaagccga caaccttgat
     2041 tggagacttg accaaacctc tggcgaagaa ttgttaatta agagctctta agacccactt
     2101 tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa taagaaggct
     2161 ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg cggcatacta
     2221 tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc ttccaatacg
     2281 caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc tagtgaaaaa
     2341 ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt ttctgtaggc
     2401 cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca tctaaaactt
     2461 ttagcgttat tacgtaaaaa atcttgccag ctttcccctt ctaaagggca aaagtgagta
     2521 tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt attttttaca
     2581 tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg ggttgttaaa
     2641 ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt acttttatct
     2701 aatctagaca ttttaataac tagtaaaact tagattagat tgctatgctt tctttctaat
     2761 gagcaagaag taaaaaaagt tgtaatagaa caagaaaaat gaaactgaaa cttgagaaat
     2821 tgaagaccgt ttattaactt aaatatcaat gggaggtcat cgaaagagaa aaaaatcaaa
     2881 aaaaaaattt tcaagaaaaa gaaacgtgat aaaaattttt attgcctttt tcgacgaaga
     2941 aaaagaaacg aggcggtctc ttttttcttt tccaaacctt tagtacgggt aattaacgac
     3001 accctagagg aagaaagagg ggaaatttag tatgctgtgc ttgggtgttt tgaagtggta
     3061 cggcgatgcg cggagtccga gaaaatctgg aagagtaaaa aaggagtaga aacattttga
     3121 agctatggtg tggcggccgc atcttgactg atttttccat ggagggcaca gttaagccgc
     3181 taaaggcatt atccgccaag tacaattttt tactcttcga agacagaaaa tttgctgaca
     3241 ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca gaatgggcag
     3301 acattacgaa tgcacacggt gtggtgggcc caggtattgt tagcggtttg aagcaggcgg
     3361 cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt agcagaattg tcatgcaagg
     3421 gctccctatc tactggagaa tatactaagg gtactgttga cattgcgaag agcgacaaag
     3481 attttgttat cggctttatt gctcaaagag acatgggtgg aagagatgaa ggttacgatt
     3541 ggttgattat gacacccggt gtgggtttag atgacaaggg agacgcattg ggtcaacagt
     3601 atagaaccgt ggatgatgtg gtctctacag gaattcgaag tacggattag aagccgccga
     3661 gcgggtgaca gccctccgaa ggaagactct cctccgtgcg tcctcgtctt caccggtcgc
     3721 gttcctgaaa cgcagatgtg cctcgcgccg cactgctccg aacaataaag attctacaat
     3781 actagctttt atggttatga agaggaaaaa ttggcagtaa cctggcccca caaaccttca
     3841 aatgaacgaa tcaaattaac aaccatagga tgataatgcg attagttttt tagccttatt
     3901 tctggggtac tgcagcagcg aagcgatgat ttttgatcta ttaacagata tataaatgca
     3961 aaaactgtcc ctatcagtga tagagatctc cctatcagtg atagagatat tacttcttat
     4021 tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata cctctatact ttaacgtcaa
     4081 ggagaaaaac cccggatcca cgattaaaag aatgtctaga gccccaaaga agaagagaaa
     4141 agttagaccc ggggataaga aatactctat tggtttggct atcggtacaa actctgttgg
     4201 ttgggctgtt attactgatg aatacaaggt tccatccaag aagttcaagg ttttgggtaa
     4261 cactgataga cactccatca aaaagaactt gattggtgcc ttgttgttcg attctggtga
     4321 aactgctgaa gctactagat tgaaaagaac cgctagaaga agatacacca gaagaaagaa
     4381 cagaatctgc tacttgcaag aaatcttctc caacgaaatg gccaaggttg atgattcatt
     4441 cttccacaga ttggaagaat ccttcttggt cgaagaagat aagaagcacg aaagacatcc
     4501 aatcttcggt aacatcgttg atgaagttgc ttaccacgaa aagtacccaa ctatctacca
     4561 tttgagaaag aagttggttg actctaccga taaggctgat ttgagattga tctatttggc
     4621 tttggcccac atgattaagt tcagaggtca tttcttgatc gaaggtgatt tgaacccaga
     4681 taactccgat gttgataagt tgttcatcca attagtccaa acctacaatc aattattcga
     4741 agaaaaccca atcaacgcct ctggtgttga tgctaaagct attttgtctg ccagattgtc
     4801 caagtccaga agattagaaa atttgatcgc ccaattacca ggtgaaaaga agaatggttt
     4861 gttcggtaat ttgattgcct tgtctttggg tttgactcca aacttcaagt ccaatttcga
     4921 tttggctgaa gatgccaagt tgcaattatc taaggatacc tacgatgacg atttggataa
     4981 cttgttggct caaatcggtg atcaatacgc tgatttgttt ttggctgcta agaacttgtc
     5041 cgatgccatt ttgttgtccg atattttgag agtcaacacc gaaattacta aggctccatt
     5101 gtctgcctct atgatcaaaa gatacgatga acaccaccaa gacttgactt tgttgaaggc
     5161 tttggtcaga caacaattac ctgaaaagta caaagaaatt ttcttcgatc aatccaagaa
     5221 cggttacgcc ggttatattg atggtggtgc ttctcaagaa gaattttaca agttcatcaa
     5281 gccaatcttg gaaaagatgg acggtactga agaattattg gtcaagttga acagagaaga
     5341 tttgttgaga aagcaaagaa ccttcgacaa cggttctatt ccacatcaaa ttcacttggg
     5401 tgaattgcac gcaattttga gaagacaaga agatttttat ccattcttga aggacaacag
     5461 agaaaagatc gaaaagattc tgaccttcag aatcccttac tacgttggtc cattggctag
     5521 aggtaattca agatttgcct ggatgactag aaagtccgaa gaaactatta ctccttggaa
     5581 cttcgaagaa gttgtagata agggtgcttc tgcccaatcc tttattgaaa gaatgaccaa
     5641 cttcgacaag aacttgccaa acgaaaaggt tttgccaaag cactctttgt tgtacgaata
     5701 cttcaccgtc tacaacgaat tgactaaggt taagtacgtc accgaaggta tgagaaaacc
     5761 agctttttta tccggtgaac aaaagaaggc tatcgtcgat ttgttgttca agaccaacag
     5821 aaaggttact gtcaagcaat taaaagaaga ttacttcaag aaaatcgaat gcttcgactc
     5881 cgttgaaatt tctggtgtcg aagatagatt caatgcctct ttaggtactt accatgactt
     5941 gttgaaaatc atcaaggaca aggatttctt ggacaacgaa gaaaacgaag atattttgga
     6001 agatattgtc ttgacattga ccttgtttga agatagagaa atgattgaag aaagattgaa
     6061 aacctacgcc cacttgttcg atgataaggt tatgaagcaa ttaaagagaa gaagatacac
     6121 tggttggggt agattgtcca gaaaattgat taacggtatc agagacaagc aatccggtaa
     6181 gaccattttg gactttttga agtctgatgg tttcgctaac agaaacttca tgcaattaat
     6241 ccacgacgat tccttgactt tcaaagaaga tatacaaaag gcccaagtct ctggtcaagg
     6301 tgattcttta catgaacata tcgctaactt ggctggttct ccagctatta agaagggtat
     6361 tttacaaacc gttaaggtcg ttgacgaatt ggtcaaagtt atgggtagac ataagccaga
     6421 aaacatcgtt atcgaaatgg ctagagaaaa tcaaaccacc caaaagggtc aaaagaactc
     6481 cagagaaaga atgaagagaa tcgaagaagg tatcaaagaa ttgggttccc aaattttgaa
     6541 agaacaccca gttgaaaaca cccaattaca aaacgaaaag ttgtacttgt actacttgca
     6601 aaacggtaga gatatgtacg ttgaccaaga attggacatc aacagattgt ctgattacga
     6661 tgttgacgct atcgttccac aatctttttt gaaggatgac tccattgaca acaaggtctt
     6721 gactagatcc gataagaata gaggtaagtc cgataacgtt ccatctgaag aagtcgttaa
     6781 gaaaatgaag aactattgga gacaattatt gaacgccaag ttgatcaccc aaagaaagtt
     6841 tgacaatttg accaaggctg aaagaggtgg tttgtctgaa ttggataagg caggtttcat
     6901 caaaagacaa ttagtagaaa ccagacaaat caccaagcac gttgctcaaa ttttggatag
     6961 tagaatgaac actaagtacg acgaaaacga caaattgatc agagaagtta aggtcattac
     7021 cttgaagtcc aagttggttt ccgatttcag aaaggacttc caattctaca aggtcagaga
     7081 aatcaacaac taccatcatg cacatgatgc ttacttgaat gctgttgttg gtactgcctt
     7141 gattaagaag tatccaaagt tggaatccga atttgtctac ggtgattaca aggtttacga
     7201 cgttagaaag atgatcgcca agtccgaaca agaaattggt aaagctactg ccaaatactt
     7261 cttctactcc aatattatga atttctttaa gaccgaaatc actttggcca acggtgaaat
     7321 tagaaaaaga ccattgattg aaactaatgg tgaaacaggt gaaatcgttt gggataaggg
     7381 tagagatttt gccactgtta gaaaggtatt gtccatgcca caagtaaaca tcgtcaaaaa
     7441 gaccgaagtt caaactggtg gtttctccaa agaatccatt ttgcctaaga gaaactccga
     7501 taagttgatc gctagaaaaa aagactggga cccaaaaaag tacggtggtt ttgattctcc
     7561 aactgttgct tactctgttt tggttgttgc taaggtcgaa aagggtaaga gtaagaagtt
     7621 gaagtccgtc aaagaattat taggtatcac tatcatggaa agatcctcat tcgaaaagaa
     7681 tcctatcgac tttttggaag ccaagggtta caaagaagtc aagaaggact tgatcattaa
     7741 gttgccaaag tacagtttgt tcgaattgga aaatggtaga aagagaatgt tggcttctgc
     7801 cggtgaatta caaaagggta atgaattggc tttgccatcc aagtacgtta atttcttata
     7861 cttggcctcc cactacgaaa aattgaaagg ttctcctgaa gataacgaac aaaagcaatt
     7921 atttgtcgaa caacacaagc actacttgga cgaaatcatt gaacaaattt ccgaattttc
     7981 caaaagagtc attttggctg acgccaattt ggacaaagtt ttgtcagctt acaacaagca
     8041 cagagataag ccaattagag aacaagctga aaacatcatt cacttgttca ctttgactaa
     8101 cttgggtgct ccagctgctt ttaagtattt cgataccact atcgacagaa agagatacac
     8161 ctctaccaaa gaagttttgg acgctacttt gatccaccaa tctattactg gtttgtacga
     8221 aactagaatc gacttgtctc aattaggtgg tgattaatag gtcgacggtc ctaggaactc
     8281 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     8341 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     8401 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     8461 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
     8521 ctttaatttg cggcccggta cccagctttt gttcccttta gtgagggtta attccgagct
     8581 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     8641 acaacatagg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgaggtaac
     8701 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     8761 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     8821 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     8881 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     8941 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     9001 ataggctcgg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     9061 acccgacagg actataaaga taccaggcgt tcccccctgg aagctccctc gtgcgctctc
     9121 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     9181 cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     9241 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     9301 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     9361 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     9421 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     9481 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     9541 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     9601 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     9661 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     9721 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     9781 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactgccc gtcgtgtaga
     9841 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     9901 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     9961 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
    10021 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
    10081 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
    10141 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
    10201 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
    10261 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
    10321 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
    10381 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
    10441 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
    10501 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
    10561 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
    10621 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
    10681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
    10741 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
    10801 cgaggccctt tcgtc
//
