LOCUS       sequence-397643-        9987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses S. Pyogenes dCas9 fused to SmallBiT fragment of split 
            NanoLuc luciferase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9987)
  AUTHORS   van der Veer HJ, van Aalen EA, Michielsen CMS, Hanckmann ETL, 
            Deckers J, van Borren MMGJ, Flipse J, Loonen AJM, Schoeber JPH, 
            Merkx M
  TITLE     Glow-in-the-Dark Infectious Disease Diagnostics Using 
            CRISPR-Cas9-Based Split Luciferase Complementation.
  JOURNAL   ACS Cent Sci. 2023 Mar 15;9(4):657-667. doi: 
            10.1021/acscentsci.2c01467. eCollection 2023 Apr 26.
  PUBMED    37122471
REFERENCE   2  (bases 1 to 9987)
  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/acscentsci.2c01467"; journalName: "ACS Cent Sci"; date: 
            "2023-03-15- 15"; volume: "9"; issue: "4"; pages: "657-667"
COMMENT     Sequence Label: pET28a(+)-SpdCas9-SBiT_Strep-Tag-II
FEATURES             Location/Qualifiers
     source          1..9987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      24..479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(111..130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     321..342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(571..1386)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(571..1386)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1294..1313
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1378..1397
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1508..2096
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1997..2016
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2250..2267
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2282..2421
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2347..2366)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2507..2529
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2523..2714)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3523..4605)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4567..4586
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4606..4683)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4889..4908
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4992..5011
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4992..5010
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5011..5035
                     /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)."
     RBS             5050..5072
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(5079..5105,5106..5108,5109..7595,7596..7598,
                     7599..9182)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             9294..9317
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             9810..9833
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             9843..9860
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(9913..9931)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      9927..9974
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
       61 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
      121 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
      181 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
      241 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
      301 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
      361 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
      421 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
      481 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
      541 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
      601 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
      661 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
      721 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
      781 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
      841 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
      901 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
      961 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     1021 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     1081 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     1141 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     1201 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     1261 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     1321 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     1381 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     1441 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1501 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1561 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1621 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1681 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1741 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1801 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1861 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1921 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1981 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2041 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2101 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2161 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2221 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2281 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     2341 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     2401 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2461 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     2521 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     2581 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     2641 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     2701 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     2761 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     2821 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     2881 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     2941 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     3001 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     3061 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     3121 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     3181 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     3241 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     3301 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     3361 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     3421 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     3481 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     3541 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3601 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     3661 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     3721 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     3781 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     3841 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     3901 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     3961 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4021 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4081 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     4141 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4201 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4261 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     4321 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     4381 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     4441 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     4501 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     4561 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     4621 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     4681 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     4741 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     4801 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     4861 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     4921 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     4981 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     5041 agaaataatt ttgtttaact ttaagaagga gatataccat ggataagaaa tactcaatag
     5101 gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa tataaggttc
     5161 cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa aaaaatctta
     5221 taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc aaacggacag
     5281 ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag attttttcaa
     5341 atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct tttttggtgg
     5401 aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat gaagttgctt
     5461 atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat tctactgata
     5521 aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt cgtggtcatt
     5581 ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta tttatccagt
     5641 tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt ggagtagatg
     5701 ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat ctcattgctc
     5761 agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg tcattgggtt
     5821 tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta cagctttcaa
     5881 aagatactta cgatgatgat ttagataatt tattggcgca aattggagat caatatgctg
     5941 atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat atcctaagag
     6001 taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc tacgatgaac
     6061 atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca gaaaagtata
     6121 aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat gggggagcta
     6181 gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat ggtactgagg
     6241 aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg
     6301 gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga agacaagaag
     6361 acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg acttttcgaa
     6421 ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga
     6481 agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa ggtgcttcag
     6541 ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat gaaaaagtac
     6601 taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg acaaaggtca
     6661 aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag aagaaagcca
     6721 ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta aaagaagatt
     6781 atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa gatagattta
     6841 atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa gattttttgg
     6901 ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc ttatttgaag
     6961 atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat gataaggtga
     7021 tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga aaattgatta
     7081 atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa tcagatggtt
     7141 ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt aaagaagaca
     7201 ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt gcaaatttag
     7261 ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt gatgaattgg
     7321 tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca cgtgaaaatc
     7381 agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta
     7441 tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact caattgcaaa
     7501 atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg gaccaagaat
     7561 tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa agtttcctta
     7621 aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt ggtaaatcgg
     7681 ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga caacttctaa
     7741 acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt
     7801 tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact cgccaaatca
     7861 ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat gaaaatgata
     7921 aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct gacttccgaa
     7981 aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt
     8041 atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt gaatcggagt
     8101 ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag tctgagcaag
     8161 aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac ttcttcaaaa
     8221 cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa actaatgggg
     8281 aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc aaagtattgt
     8341 ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg
     8401 agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa gactgggatc
     8461 caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta gtggttgcta
     8521 aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta gggatcacaa
     8581 ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct aaaggatata
     8641 aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt gagttagaaa
     8701 acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat gagctggctc
     8761 tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag ttgaagggta
     8821 gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat tatttagatg
     8881 agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat gccaatttag
     8941 ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa caagcagaaa
     9001 atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt aaatattttg
     9061 atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat gccactctta
     9121 tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag ctaggaggtg
     9181 acaccggtgg gggtagcggc ggctcggggg gtagtggtgg aagcgggggt tcaaagctta
     9241 ctagtgttac cggctatcgt ctgtttgaaa aagagagcgg atccggtgga agctggagcc
     9301 atccgcagtt tgaaaaataa actagtgtct tcacactcga agatttcgtt ggggactggg
     9361 aacagacagc cgcctacaac ctggaccaag tccttgaaca gggaggtgtg tccagtttgc
     9421 tgcagaatct cgccgtgtcc gtaactccga tccaaaggat tgtccggagc ggtgaaaatg
     9481 ccctgaagat cgacatccat gtcatcatcc cgtatgaagg tctgagcgcc gaccaaatgg
     9541 cccagatcga agaggtgttt aaggtggtgt accctgtgga tgatcatcac tttaaggtga
     9601 tcctgcccta tggcacactg gtaatcgacg gggttacgcc gaacatgctg aactatttcg
     9661 gacggccgta tgaaggcatc gccgtgttcg acggcaaaaa gatcactgta acagggaccc
     9721 tgtggaacgg caacaaaatt atcgacgagc gcctgatcac ccccgacggc tccatgctgt
     9781 tccgagtaac catcaacagc ggtggaagct ggagccatcc gcagtttgaa aaataactcg
     9841 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     9901 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     9961 tgaggggttt tttgctgaaa ggaggaa
//
