LOCUS       sequence-350706-       10178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid to express Cas9 from S.pyogenesand CRISPR gRNA to introduce 
            LRRK2-G2019S mutation in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10178)
  AUTHORS   Li H, Busquets O, Verma Y, Syed KM, Kutnowski N, Pangilinan GR, 
            Gilbert LA, Bateup HS, Rio DC, Hockemeyer D, Soldner F
  TITLE     Highly efficient generation of isogenic pluripotent stem cell models
            using prime editing.
  JOURNAL   Elife. 2022 Sep 7;11:e79208. doi: 10.7554/eLife.79208.
  PUBMED    36069759
REFERENCE   2  (bases 1 to 10178)
  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: "2022-09-7"; volume: "11:e79208. doi"; pages: " 
            10.7554/eLife.79208"
COMMENT     Sequence Label: Px330-EGFP-LRRK2-CRISPR/Cas9
FEATURES             Location/Qualifiers
     source          1..10178
                     /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"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        439..742
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        744..1021
                     /label=chicken beta-actin promoter
     intron          1022..1250
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1262..1271
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1271..1336
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1343..1363
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1388..5488
                     /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             1388..5488
                     /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             5489..5536
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5564..5581)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5570..5777
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5846..6149
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6150..6353
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6304..6324
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(6382..6384,6385..6387,6388..7101)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6382..6384,6385..6387,6388..7098)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6688..6707)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7035..7056
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      7656..8111
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7743..7762)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7953..7974
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8128..8147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8247..8269
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8307..8325)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8393..8497
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8498..8566,8567..9358)
                     /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(8716..8735)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9529..10117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10018..10037
                     /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 attgcaaaga ttgctgacta gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgtagcgc gtgcgccaat tctgcagaca
      421 aatggctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      481 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      541 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      601 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      661 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      721 attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat
      781 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      841 gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      901 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      961 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
     1021 gggagtcgct gcgacgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1081 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1141 ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg
     1201 tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg
     1261 tgccaccatg gactataagg accacgacgg agactacaag gatcatgata ttgattacaa
     1321 agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc
     1381 agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc
     1441 cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga
     1501 ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc
     1561 cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat
     1621 ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca
     1681 cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt
     1741 cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag
     1801 aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc
     1861 ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag
     1921 cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa
     1981 ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag
     2041 cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg
     2101 caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc
     2161 cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct
     2221 ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc
     2281 catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc
     2341 ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt
     2401 gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta
     2461 cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat
     2521 cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct
     2581 gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct
     2641 gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa
     2701 gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa
     2761 cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga
     2821 ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga
     2881 taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac
     2941 cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt
     3001 cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt
     3061 gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga
     3121 aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa
     3181 aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat
     3241 cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta
     3301 tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg
     3361 gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat
     3421 cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga
     3481 cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag
     3541 cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca
     3601 gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat
     3661 cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga
     3721 gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca
     3781 ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg
     3841 gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga
     3901 ccatatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag
     3961 aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat
     4021 gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa
     4081 tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag
     4141 acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat
     4201 gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa
     4261 gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa
     4321 caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa
     4381 aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg
     4441 gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta
     4501 cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa
     4561 gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga
     4621 ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga
     4681 ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct
     4741 gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt
     4801 ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag
     4861 tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat
     4921 cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc
     4981 taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga
     5041 actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc
     5101 cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt
     5161 ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag
     5221 agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga
     5281 taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg
     5341 agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac
     5401 caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg
     5461 gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca
     5521 ggcaaaaaag aaaaagtaag aattcctaga gctcgctgat cagcctcgac tgtgccttct
     5581 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     5641 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     5701 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     5761 agcaggcatg ctggggagcg gccgctagtt attaatagta atcaattacg gggtcattag
     5821 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     5881 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     5941 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     6001 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     6061 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     6121 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     6181 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     6241 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     6301 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     6361 tgaaccgtca gatccgctag catggtgagc aagggcgagg agctgttcac cggggtggtg
     6421 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     6481 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     6541 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     6601 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     6661 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     6721 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     6781 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     6841 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     6901 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     6961 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     7021 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     7081 atggacgagc tgtacaagta aggatccggg atgcagaaat tgatgatcta ttaaacaata
     7141 aagatgtcca ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag
     7201 agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag
     7261 ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt
     7321 tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca
     7381 tgatctatag atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg
     7441 tggttctaag tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag
     7501 cagcctctgt tccacataca cttcattctc agtattgttt tgccaagttc taattccatc
     7561 agaagctggt cgacctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     7621 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     7681 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     7741 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     7801 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     7861 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     7921 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     7981 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     8041 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     8101 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     8161 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     8221 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     8281 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     8341 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     8401 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     8461 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     8521 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     8581 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     8641 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     8701 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     8761 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     8821 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     8881 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     8941 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     9001 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     9061 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     9121 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     9181 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     9241 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     9301 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     9361 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     9421 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     9481 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     9541 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     9601 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     9661 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     9721 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     9781 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     9841 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     9901 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     9961 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
    10021 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
    10081 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
    10141 acggttcctg gccttttgct ggccttttgc tcacatgt
//
