LOCUS       sequence-339962-        9926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR interference. Construct for expressing catalytically inactive
            Cas9 (dCas9) from Streptococcus pyogenes in Toxoplasma gondii. 
            Suitable for generating stable cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9926)
  AUTHORS   Markus BM, Boydston EA, Lourido S
  TITLE     CRISPR-Mediated Transcriptional Repression in Toxoplasma gondii.
  JOURNAL   mSphere. 2021 Oct 13:e0047421. doi: 10.1128/mSphere.00474-21.
  PUBMED    34643425
REFERENCE   2  (bases 1 to 9926)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "mSphere. 
            2021 Oct 13:e0047421. doi: 10.1128/mSphere.00474-21."
COMMENT     Sequence Label: pTUB1-Spy.dCas9-CAT-U6-sgRNA(decoy)
FEATURES             Location/Qualifiers
     source          1..9926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   144..284
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   155..284
                     /label=AAV2 ITR
     polyA_signal    293..500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     489..506
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(534..581)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(join(582..2165,2166..2168,2169..4655,4656..4658,
                     4659..4682))
                     /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"
     CDS             complement(582..4682)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(4707..4727)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4734..4799)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_RNA        complement(5330..5405)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5933..5949
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5933..5949
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             complement(6267..6929)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MHEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF
                     LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFS
                     SLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVAN
                     MDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6841..6860
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(7444..7460)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7445..7461)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     rep_origin      complement(7534..8122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7614..7633)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8293..9084,9085..9153))
                     /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     8916..8935
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9154..9258)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     9326..9344
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(9382..9404)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9504..9523
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(9540..9926,1..69))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9677..9698)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     9889..9908
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
       61 agggcgcgta ctatggttgc tttgacgtat gcggtgtgaa ataccgcaca gatgcgtaag
      121 gagaaaatac cgcatcaggc gcccctgcag gcagctgcgc gctcgctcgc tcactgaggc
      181 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
      241 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gctccccagc
      301 atgcctgcta ttctcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
      361 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
      421 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
      481 ctggcaacta gaaggcacag tcgaggctga tcagcgagct ctaggaattc ttactttttc
      541 ttttttgcct ggccggcctt tttcgtggcc gccggccttt tgtcgcctcc cagctgagac
      601 aggtcgatcc gtgtctcgta caggccggtg atgctctggt ggatcagggt ggcgtccagc
      661 acctctttgg tgctggtgta cctcttccgg tcgatggtgg tgtcaaagta cttgaaggcg
      721 gcaggggctc ccagattggt cagggtaaac aggtggatga tattctcggc ctgctctctg
      781 atgggcttat cccggtgctt gttgtaggcg gacagcactt tgtccagatt agcgtcggcc
      841 aggatcactc tcttggagaa ctcgctgatc tgctcgatga tctcgtccag gtagtgcttg
      901 tgctgttcca caaacagctg tttctgctca ttatcctcgg gggagccctt cagcttctca
      961 tagtggctgg ccaggtacag gaagttcaca tatttggagg gcagggccag ttcgtttccc
     1021 ttctgcagtt cgccggcaga ggccagcatt ctcttccggc cgttttccag ctcgaacagg
     1081 gagtacttag gcagcttgat gatcaggtcc tttttcactt ctttgtagcc cttggcttcc
     1141 agaaagtcga tgggattctt ctcgaagctg cttctttcca tgatggtgat ccccagcagc
     1201 tctttcacac tcttcagttt cttggacttg cccttttcca ctttggccac caccagcaca
     1261 gaataggcca cggtggggct gtcgaagccg ccgtacttct tagggtccca gtccttcttt
     1321 ctggcgatca gcttatcgct gttcctcttg ggcaggatag actctttgct gaagccgcct
     1381 gtctgcacct cggtcttttt cacgatattc acttggggca tgctcagcac tttccgcacg
     1441 gtggcaaaat cccggccctt atcccacacg atctccccgg tttcgccgtt tgtctcgatc
     1501 agaggccgct tccggatctc gccgttggcc agggtaatct cggtcttgaa aaagttcatg
     1561 atgttgctgt agaagaagta cttggcggta gccttgccga tttcctgctc gctcttggcg
     1621 atcatcttcc gcacgtcgta caccttgtag tcgccgtaca cgaactcgct ttccagctta
     1681 gggtactttt tgatcagggc ggttcccacg acggcgttca ggtaggcgtc gtgggcgtgg
     1741 tggtagttgt tgatctcgcg cactttgtaa aactggaaat ccttccggaa atcggacacc
     1801 agcttggact tcagggtgat cactttcact tcccggatca gcttgtcatt ctcgtcgtac
     1861 ttagtgttca tccgggagtc caggatctgt gccacgtgct ttgtgatctg ccgggtttcc
     1921 accagctgtc tcttgatgaa gccggcctta tccagttcgc tcaggccgcc tctctcggcc
     1981 ttggtcagat tgtcgaactt tctctgggta atcagcttgg cgttcagcag ctgccgccag
     2041 tagttcttca tcttcttcac gacctcttcg gagggcacgt tgtcgctctt gccccggttc
     2101 ttgtcgcttc tggtcagcac cttgttgtcg atggagtcgt ccttcagaaa gctctgaggc
     2161 acgatagcgt ccacatcgta gtcggacagc cggttgatgt ccagttcctg gtccacgtac
     2221 atatcccgcc cattctgcag gtagtacagg tacagcttct cgttctgcag ctgggtgttt
     2281 tccacggggt gttctttcag gatctggctg cccagctctt tgatgccctc ttcgatccgc
     2341 ttcattctct cgcggctgtt cttctgtccc ttctgggtgg tctggttctc tctggccatt
     2401 tcgatcacga tgttctcggg cttgtgccgg cccatcactt tcacgagctc gtccaccacc
     2461 ttcactgtct gcaggatgcc cttcttaatg gcggggctgc cggccagatt ggcaatgtgc
     2521 tcgtgcaggc tatcgccctg gccggacacc tgggctttct ggatgtcctc tttaaaggtc
     2581 aggctgtcgt cgtggatcag ctgcatgaag tttctgttgg cgaagccgtc ggacttcagg
     2641 aaatccagga ttgtcttgcc ggactgcttg tcccggatgc cgttgatcag cttccggctc
     2701 agcctgcccc agccggtgta tctccgccgc ttcagctgct tcatcacttt gtcgtcgaac
     2761 aggtgggcat aggttttcag ccgttcctcg atcatctctc tgtcctcaaa cagtgtcagg
     2821 gtcagcacga tatcttccag aatgtcctcg ttttcctcat tgtccaggaa gtccttgtcc
     2881 ttgataattt tcagcagatc gtggtatgtg cccagggagg cgttgaaccg atcttccacg
     2941 ccggagattt ccacggagtc gaagcactcg attttcttga agtagtcctc tttcagctgc
     3001 ttcacggtca ctttccggtt ggtcttgaac agcaggtcca cgatggcctt tttctgctcg
     3061 ccgctcagga aggcgggctt tctcattccc tcggtcacgt atttcacttt ggtcagctcg
     3121 ttatacacgg tgaagtactc gtacagcagg ctgtgcttgg gcagcacctt ctcgttgggc
     3181 aggttcttat cgaagttggt catccgctcg atgaagctct gggcggaagc gcccttgtcc
     3241 accacttcct cgaagttcca gggggtgatg gtttcctcgc tctttctggt catccaggcg
     3301 aatctgctgt ttcccctggc cagagggccc acgtagtagg ggatgcggaa ggtcaggatc
     3361 ttctcgatct tttcccggtt gtccttcagg aatgggtaaa aatcttcctg ccgccgcaga
     3421 atggcgtgca gctctcccag gtggatctgg tgggggatgc tgccgttgtc gaaggtccgc
     3481 tgcttccgca gcaggtcctc tctgttcagc ttcacgagca gttcctcggt gccgtccatc
     3541 ttttccagga tgggcttgat gaacttgtag aactcttcct ggctggctcc gccgtcaatg
     3601 tagccggcgt agccgttctt gctctggtcg aagaaaatct ctttgtactt ctcaggcagc
     3661 tgctgccgca cgagagcttt cagcagggtc aggtcctggt ggtgctcgtc gtatctcttg
     3721 atcatagagg cgctcagggg ggccttggtg atctcggtgt tcactctcag gatgtcgctc
     3781 agcaggatgg cgtcggacag gttcttggcg gccagaaaca ggtcggcgta ctggtcgccg
     3841 atctgggcca gcaggttgtc caggtcgtcg tcgtaggtgt ccttgctcag ctgcagtttg
     3901 gcatcctcgg ccaggtcgaa gttgctcttg aagttggggg tcaggcccag gctcagggca
     3961 atcaggtttc cgaacaggcc attcttcttc tcgccgggca gctgggcgat cagattttcc
     4021 agccgtctgc tcttgctcag tctggcagac aggatggcct tggcgtccac gccgctggcg
     4081 ttgatggggt tttcctcgaa cagctggttg taggtctgca ccagctggat gaacagcttg
     4141 tccacgtcgc tgttgtcggg gttcaggtcg ccctcgatca ggaagtggcc ccggaacttg
     4201 atcatgtggg ccagggccag atagatcagc cgcaggtcgg ccttgtcggt gctgtccacc
     4261 agtttctttc tcaggtggta gatggtgggg tacttctcgt ggtaggccac ctcgtccacg
     4321 atgttgccga agatggggtg ccgctcgtgc ttcttatcct cttccaccag gaaggactct
     4381 tccagtctgt ggaagaagct gtcgtccacc ttggccatct cgttgctgaa gatctcttgc
     4441 agatagcaga tccggttctt ccgtctggtg tatcttcttc tggcggttct cttcagccgg
     4501 gtggcctcgg ctgtttcgcc gctgtcgaac agcagggctc cgatcaggtt cttcttgatg
     4561 ctgtgccggt cggtgttgcc cagcaccttg aatttcttgc tgggcacctt gtactcgtcg
     4621 gtgatcacgg cccagcccac agagttggtg ccgatggcca ggccgatgct gtacttcttg
     4681 tcggctgctg ggactccgtg gataccgacc ttccgcttct tctttggggc catcttatcg
     4741 tcatcgtctt tgtaatcaat atcatgatcc ttgtagtctc cgtcgtggtc cttatagtcc
     4801 atggtgtcga aaaagggaat tcaagaaaaa atgccaacga gtagttttcc gcaagaactt
     4861 cacacaaggt gattgtgtaa caccgtttgg tggatgtctt ctgcgcgggt tgcgtcgtca
     4921 gggacggcga acgagtgctt gttctctacg agactcgcaa agagaaggaa agggcgaaat
     4981 tcaagagaga atccaacaag gaggactctc tgctaatatt cgccgccttt aacacatcta
     5041 aaagttcaca gagaacaaac aaaatgtttc tgacacatcc ccttcgtgcg cgcgcatctc
     5101 caccggtcca accaggatca ccaaccgcac ccaatgcagg caccacccct gttctgcgat
     5161 gagcgcaagc cgtcctgagt tacgccttcg gcatctgact gcccaccgcg agccggcctg
     5221 ctccgcaacg tcatcccgct gagacgatga ttcctgatca ctccgctgat gcagagaatt
     5281 tcacgaacgc gggacatgca tggtacctct agagaaaaag aaaaaaaaag caccgactcg
     5341 gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat ttctagctct
     5401 aaaacacggt gctaaactgc attctcaact tgacatcccc atttaccaga aggcaaacac
     5461 ccccttcggg gacgaggtga ccctgcgcga cagaaagccc cttcgaagag cgcacaggga
     5521 ggaagcaggc ctctgcaggt cgccatttga aaatctgaca gaactgatgg aaatatgatt
     5581 cttgtcagag aagacattcg agagttcgaa ggtttccccc ttggctctac atatcccagt
     5641 gtctcgcgtt ctgcaggagg cgcgtcaggc ctaggatgca atattggcgc ccaattcaca
     5701 gtgcagcggc gcagccgtcg caacacttcg cagcgtgaaa gacttatcgc tctccgcctc
     5761 tcgatggttc gggatgttcg cttcgagtgt atgctgcgca agctttcgcg tttgggtacg
     5821 ttaatttgaa atctttcgcg tttcccgtgt gccctcagtg tctccaacat gtggccgcgg
     5881 tgaaatcgtc cccacgacga tttcaactcg cgcactttgt ctcatcggta cctctagaac
     5941 tagtggatcc ccctcggggg ggcaagaatt gtgttaaccg gttcgactaa aacaactgat
     6001 agctgtcttg tgtattgacc catgtggcgc gtgtgtcgac ggcgatcaac atcagccagg
     6061 tccgccacac atccgttcag agattcggac ccagtcataa actttcgctg cttgctgcga
     6121 aacactccta tttagagtcg gccggttttt gtggatgtga cacctgcaag ccacaacgga
     6181 acaactcagt ttctttataa tggggcacat gctgcacgaa gtgtgtttcc tttgtcgatt
     6241 tgagaagtga gcacaacggt gattaattaa gccccgccct gccactcatc gcagtactgt
     6301 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     6361 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     6421 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     6481 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     6541 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     6601 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     6661 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga
     6721 gcattcatca ggcgggcaag aatgtgaata aatgccggat aaaacttgtg cttatttttc
     6781 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     6841 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     6901 gtatatccag tgattttttt ctcatgcatt ttgtcgaaaa agggaattca agaaaaaatg
     6961 ccaacgagta gttttccgca agaacttcac acaaggtgat tgtgtaacac cgtttggtgg
     7021 atgtcttctg cgcgggttgc gtcgtcaggg acggcgaacg agtgcttgtt ctctacgaga
     7081 ctcgcaaaga gaaggaaagg gcgaaattca agagagaatc caacaaggag gactctctgc
     7141 taatattcgc cgcctttaac acatctaaaa gttcacagag aacaaacaaa atgtttctga
     7201 cacatcccct tcgtgcgcgc gcatctccac cggtccaacc aggatcacca accgcaccca
     7261 atgcaggcac cacccctgtt ctgcgatgag cgcaagccgt cctgagttac gccttcggca
     7321 tctgactgcc caccgcgagc cggcctgctc cgcaacgtca tcccgctgag acgatgattc
     7381 ctgatcactc cgctgatgca gagaatttca cgaacgcggg acatgcatcg ccatcaagct
     7441 tatcgatacc gtcgacctcg agggggggcc cgacatgtga gcaaaaggcc agcaaaaggc
     7501 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7561 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7621 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7681 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7741 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7801 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7861 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7921 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7981 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     8041 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     8101 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     8161 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     8221 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     8281 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     8341 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     8401 accatctggc cccagtgctg caatgatacc gcggcttcca cgctcaccgg ctccagattt
     8461 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     8521 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     8581 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8641 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8701 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8761 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8821 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8881 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8941 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     9001 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     9061 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     9121 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     9181 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     9241 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     9301 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     9361 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     9421 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     9481 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa
     9541 aattgtaaac gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     9601 ttttaaccaa tagaccgaaa tcggcaaaat cccttataaa tcaaaagaat agcccgagat
     9661 agagttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     9721 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccaa
     9781 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     9841 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     9901 gaaaggagcg ggcgctaagg cgctgg
//
