LOCUS       sequence-147632-        9299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human codon optimized plasmid to express Cas9 t2a mCherry w/2xNLS 
            and a sgRNA .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9299)
  AUTHORS   Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, 
            Savage DF
  TITLE     Profiling of engineering hotspots identifies an allosteric 
            CRISPR-Cas9 switch.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528.
  PUBMED    27136077
REFERENCE   2  (bases 1 to 9299)
  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: "Nat 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528."
COMMENT     Sequence Label: pBLO1806_Cas9_2xNLS_human
FEATURES             Location/Qualifiers
     source          1..9299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        450..735
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        737..1014
                     /label=chicken beta-actin promoter
     intron          1015..1243
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1255..1264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1264..1329
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1336..1356
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1381..5481
                     /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             1381..5481
                     /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             5482..5529
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5551..5604
                     /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             5605..6315
                     /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(5750..5768)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(6340..6357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6346..6553
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6562..6702
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6562..6691
                     /label=AAV2 ITR
     rep_origin      6777..7232
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6864..6883)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7074..7095
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7249..7268)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7368..7390
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7428..7446)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7514..7618
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7619..7687,7688..8479)
                     /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(7837..7856)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8650..9238
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9139..9158
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt taagagctat gctggaaaca gcatagcaag
      301 tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
      361 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa
      421 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct
      481 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
      541 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
      601 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
      661 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
      721 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
      781 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
      841 gcgggggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg
      901 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
      961 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
     1021 gctgcgacgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
     1081 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
     1141 ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat
     1201 gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc
     1261 atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat
     1321 gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
     1381 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
     1441 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
     1501 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
     1561 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
     1621 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
     1681 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     1741 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     1801 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1861 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1921 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     1981 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     2041 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     2101 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     2161 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     2221 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     2281 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     2341 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     2401 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     2461 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     2521 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     2581 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     2641 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     2701 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     2761 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     2821 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     2881 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     2941 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     3001 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     3061 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     3121 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     3181 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     3241 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     3301 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     3361 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     3421 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     3481 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     3541 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     3601 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     3661 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     3721 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     3781 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3841 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     3901 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     3961 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     4021 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     4081 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     4141 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     4201 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     4261 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     4321 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     4381 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     4441 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     4501 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     4561 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     4621 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     4681 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     4741 agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat
     4801 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4861 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     4921 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     4981 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     5041 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     5101 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     5161 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     5221 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     5281 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     5341 gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag
     5401 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     5461 ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     5521 aagaaaaagg gatccgaatc cggcagtgga gagggcagag gaagtctgct aacatgcggt
     5581 gacgtcgagg agaatcctgg cccaatggtg agcaagggcg aggaggataa catggccatc
     5641 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     5701 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     5761 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     5821 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     5881 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     5941 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     6001 aacttcccct ccgacggccc cgtaatgcag aagaaaacca tgggctggga ggcctcctcc
     6061 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     6121 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     6181 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     6241 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     6301 gagctgtaca agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     6361 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     6421 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     6481 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     6541 ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     6601 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     6661 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     6721 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     6781 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6841 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6901 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6961 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     7021 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     7081 cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg
     7141 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     7201 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     7261 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     7321 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     7381 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     7441 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     7501 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     7561 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     7621 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     7681 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     7741 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     7801 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     7861 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     7921 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     7981 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     8041 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     8101 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     8161 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     8221 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     8281 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     8341 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     8401 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     8461 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     8521 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     8581 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     8641 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     8701 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     8761 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     8821 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     8881 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     8941 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     9001 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     9061 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     9121 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     9181 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     9241 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt
//
