LOCUS       sequence-330979-        9705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding for 2 gRNAs targeting the human BAK gene and a CMV 
            driven nuclease Cas9 followed by self-cleaving mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9705)
  AUTHORS   Tigano M, Vargas DC, Tremblay-Belzile S, Fu Y, Sfeir A
  TITLE     Nuclear sensing of breaks in mitochondrial DNA enhances immune 
            surveillance.
  JOURNAL   Nature. 2021 Mar;591(7850):477-481. doi: 10.1038/s41586-021-03269-w.
            Epub 2021 Feb 24.
  PUBMED    33627873
REFERENCE   2  (bases 1 to 9705)
  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: 
            "10.1038/s41586-021-03269-w"; journalName: "Nature"; date: 
            "2021-03"; volume: "591"; issue: "7850"; pages: "477-481"
COMMENT     Sequence Label: pSpCas9nucl-T2A-mCherry - BAKgRNA1- BAKgRNA2
FEATURES             Location/Qualifiers
     source          1..9705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        2..287
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        289..566
                     /label=chicken beta-actin promoter
     intron          567..795
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      807..816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             816..881
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             888..908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             933..5033
                     /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             933..5033
                     /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             5034..5081
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5088..5141
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5142..5849
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(5287..5305)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(5877..5894)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5883..6090
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6099..6239
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6099..6228
                     /label=AAV2 ITR
     rep_origin      6314..6769
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6401..6420)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6611..6632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6786..6805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6905..6927
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6965..6983)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7051..7155
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7156..7224,7225..8016)
                     /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(7374..7393)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8187..8775
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8676..8695
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8837..9085
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        9106..9181
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        9272..9520
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     misc_RNA        9541..9616
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
       61 tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      121 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      181 acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc ttatgggact
      241 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
      301 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
      361 ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag
      421 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      481 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      541 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc
      601 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      661 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
      721 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
      781 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
      841 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
      901 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
      961 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
     1021 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
     1081 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
     1141 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
     1201 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
     1261 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
     1321 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1381 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1441 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1501 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1561 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     1621 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     1681 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     1741 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     1801 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     1861 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     1921 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     1981 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     2041 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     2101 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     2161 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     2221 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     2281 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     2341 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2401 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2461 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     2521 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     2581 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     2641 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     2701 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     2761 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     2821 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     2881 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc
     2941 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     3001 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag
     3061 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     3121 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     3181 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     3241 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     3301 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3361 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3421 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3481 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3541 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     3601 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     3661 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     3721 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     3781 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     3841 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg
     3901 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg
     3961 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca
     4021 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
     4081 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
     4141 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
     4201 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
     4261 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
     4321 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
     4381 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
     4441 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
     4501 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
     4561 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
     4621 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
     4681 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
     4741 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
     4801 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
     4861 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
     4921 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
     4981 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc
     5041 cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa ggaattcgaa ggaaggggct
     5101 ctttgcttac ttgtggagat gttgaggaaa atccaggacc catggtgagc aagggcgagg
     5161 aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg
     5221 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc
     5281 agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt
     5341 cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     5401 acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg
     5461 gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg
     5521 tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aaaaccatgg
     5581 gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca
     5641 agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca
     5701 aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca
     5761 cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     5821 ccaccggcgg catggacgag ctgtacaagg aattctaact agagctcgct gatcagcctc
     5881 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     5941 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     6001 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     6061 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt
     6121 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     6181 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     6241 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     6301 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     6361 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     6421 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     6481 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     6541 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     6601 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     6661 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     6721 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     6781 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     6841 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     6901 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     6961 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     7021 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     7081 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     7141 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7201 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7261 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7321 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7381 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     7441 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     7501 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     7561 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     7621 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     7681 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     7741 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     7801 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     7861 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     7921 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     7981 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8041 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8101 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8161 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     8221 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     8281 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     8341 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     8401 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     8461 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     8521 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     8581 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     8641 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     8701 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     8761 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     8821 ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     8881 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     8941 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     9001 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     9061 tatcttgtgg aaaggacgaa acaccgcatg aagtcgacca cgaaggtttt agagctagaa
     9121 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     9181 cttttttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg
     9241 tgcgccaatt ctgcagacaa atggctctag agagggccta tttcccatga ttccttcata
     9301 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     9361 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     9421 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     9481 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggccatgctg gtagacgtgt
     9541 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
     9601 ggcaccgagt cggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag
     9661 tccgttttta gcgcgtgcgc caattctgca gacaaatggc ggtac
//
