LOCUS       sequence-334644-        8946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Cas9 and 2 guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8946)
  AUTHORS   Fang H, Bygrave AM, Roth RH, Johnson RC, Huganir RL
  TITLE     An optimized CRISPR/Cas9 approach for precise genome editing in 
            neurons.
  JOURNAL   Elife. 2021 Mar 10;10. pii: 65202. doi: 10.7554/eLife.65202.
  PUBMED    33689678
REFERENCE   2  (bases 1 to 8946)
  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: "Elife."; 
            date: "2021-03-10"; volume: "10. pii: 65202. doi"; pages: " 
            10.7554/eLife.65202"
COMMENT     Sequence Label: rGluA1 (px330:2-guideCas9)
FEATURES             Location/Qualifiers
     source          1..8946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          112..339
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      351..360
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             360..425
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             432..452
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             477..4577
                     /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
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             477..4577
                     /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
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4578..4625
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4653..4670)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4659..4866
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4875..5015
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4875..5004
                     /label=AAV2 ITR
     rep_origin      5090..5545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5177..5196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5387..5408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5562..5581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5681..5703
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5741..5759)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5827..5931
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5932..6000,6001..6792)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6150..6169)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6963..7551
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7452..7471
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        7613..7861
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        7882..7957
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        8048..8296
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        8318..8393
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8493..8778
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        join(8780..8946,1..111)
                     /label=chicken beta-actin promoter
ORIGIN
        1 aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg
       61 gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct
      121 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc
      181 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt
      241 aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt
      301 aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg
      361 actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg
      421 ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca
      481 agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg
      541 acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca
      601 tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc
      661 ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc
      721 aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag
      781 agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg
      841 tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg
      901 tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca
      961 agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca
     1021 agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg
     1081 ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg
     1141 aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg
     1201 ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca
     1261 aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg
     1321 gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga
     1381 gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca
     1441 agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc
     1501 tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca
     1561 ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga
     1621 tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc
     1681 ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc
     1741 tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga
     1801 tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg
     1861 cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg
     1921 acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc
     1981 ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg
     2041 agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg
     2101 agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc
     2161 agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg
     2221 tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg
     2281 acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc
     2341 tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt
     2401 tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga
     2461 gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc
     2521 tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga
     2581 cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc
     2641 acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg
     2701 tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa
     2761 tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc
     2821 ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa
     2881 acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt
     2941 acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc
     3001 ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga
     3061 accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact
     3121 ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg
     3181 ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg
     3241 aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt
     3301 acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg
     3361 tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc
     3421 acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta
     3481 agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg
     3541 ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca
     3601 tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga
     3661 tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg
     3721 tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag
     3781 gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa
     3841 agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg
     3901 tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc
     3961 tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg
     4021 aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc
     4081 tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg
     4141 gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg
     4201 agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca
     4261 agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg
     4321 ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca
     4381 gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg
     4441 ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc
     4501 tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt
     4561 ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga
     4621 aaaagtaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     4681 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4741 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4801 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     4861 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     4921 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     4981 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     5041 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     5101 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     5161 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     5221 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     5281 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     5341 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5401 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     5461 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5521 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     5581 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5641 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     5701 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     5761 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     5821 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5881 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     5941 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6001 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6061 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     6121 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     6181 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6241 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     6301 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     6361 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     6421 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     6481 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     6541 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     6601 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     6661 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     6721 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     6781 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     6841 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     6901 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     6961 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7021 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7081 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     7141 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7201 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7261 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7321 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7381 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7441 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7501 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     7561 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtgagggcct
     7621 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     7681 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     7741 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     7801 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     7861 cgggaagacc aaatctatgg tgttttagag ctagaaatag caagttaaaa taaggctagt
     7921 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat
     7981 agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg
     8041 ctctagcgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     8101 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     8161 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     8221 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     8281 gaaaggacga aacaccgcat gctcgtcaaa tgccacggtt ttagagctag aaatagcaag
     8341 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     8401 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa
     8461 ttctgcagac aaatggctct agaggtggta cccgttacat aacttacggt aaatggcccg
     8521 cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact
     8581 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     8641 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     8701 cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     8761 gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc
     8821 cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg
     8881 ggggcggggg gggggggggg gcgcgcgccr ggsggggsgg ggsggggsgr ggggsggggs
     8941 ggggsg
//
