LOCUS       sequence-355854-       10115 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 10115)
  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 10115)
  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..10115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        264..339
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        436..721
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          953..1180
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1192..1201
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1201..1266
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1273..1293
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1318..5418
                     /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             1318..5418
                     /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             5419..5466
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5494..5511)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5500..5707
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5776..6079
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6080..6283
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6234..6254
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(6312..6314,6315..6317,6318..7031)
                     /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(6312..6314,6315..6317,6318..7028)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6618..6637)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6965..6986
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      7586..8041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7673..7692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7883..7904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8058..8077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8177..8199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8237..8255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8323..8427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8428..8496,8497..9288)
                     /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(8646..8665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9459..10047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9948..9967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(10109..10115,1..242)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     join(10109..10115,1..14)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
       61 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
      121 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
      181 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
      241 ccgattgcaa agattgctga ctagttttag agctagaaat agcaagttaa aataaggcta
      301 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt agagctagaa
      361 atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct gcagacaaat
      421 ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
      481 cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt gacgtcaatg
      541 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      601 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg cccagtacat
      661 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      721 ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc
      781 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggc
      841 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
      901 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc
      961 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct
     1021 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg
     1081 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt
     1141 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tgccaccatg
     1201 gactataagg accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac
     1261 gataagatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccgac
     1321 aagaagtaca gcatcggcct ggacatcggc accaactctg tgggctgggc cgtgatcacc
     1381 gacgagtaca aggtgcccag caagaaattc aaggtgctgg gcaacaccga ccggcacagc
     1441 atcaagaaga acctgatcgg agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
     1501 cggctgaaga gaaccgccag aagaagatac accagacgga agaaccggat ctgctatctg
     1561 caagagatct tcagcaacga gatggccaag gtggacgaca gcttcttcca cagactggaa
     1621 gagtccttcc tggtggaaga ggataagaag cacgagcggc accccatctt cggcaacatc
     1681 gtggacgagg tggcctacca cgagaagtac cccaccatct accacctgag aaagaaactg
     1741 gtggacagca ccgacaaggc cgacctgcgg ctgatctatc tggccctggc ccacatgatc
     1801 aagttccggg gccacttcct gatcgagggc gacctgaacc ccgacaacag cgacgtggac
     1861 aagctgttca tccagctggt gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
     1921 gccagcggcg tggacgccaa ggccatcctg tctgccagac tgagcaagag cagacggctg
     1981 gaaaatctga tcgcccagct gcccggcgag aagaagaatg gcctgttcgg aaacctgatt
     2041 gccctgagcc tgggcctgac ccccaacttc aagagcaact tcgacctggc cgaggatgcc
     2101 aaactgcagc tgagcaagga cacctacgac gacgacctgg acaacctgct ggcccagatc
     2161 ggcgaccagt acgccgacct gtttctggcc gccaagaacc tgtccgacgc catcctgctg
     2221 agcgacatcc tgagagtgaa caccgagatc accaaggccc ccctgagcgc ctctatgatc
     2281 aagagatacg acgagcacca ccaggacctg accctgctga aagctctcgt gcggcagcag
     2341 ctgcctgaga agtacaaaga gattttcttc gaccagagca agaacggcta cgccggctac
     2401 attgacggcg gagccagcca ggaagagttc tacaagttca tcaagcccat cctggaaaag
     2461 atggacggca ccgaggaact gctcgtgaag ctgaacagag aggacctgct gcggaagcag
     2521 cggaccttcg acaacggcag catcccccac cagatccacc tgggagagct gcacgccatt
     2581 ctgcggcggc aggaagattt ttacccattc ctgaaggaca accgggaaaa gatcgagaag
     2641 atcctgacct tccgcatccc ctactacgtg ggccctctgg ccaggggaaa cagcagattc
     2701 gcctggatga ccagaaagag cgaggaaacc atcaccccct ggaacttcga ggaagtggtg
     2761 gacaagggcg cttccgccca gagcttcatc gagcggatga ccaacttcga taagaacctg
     2821 cccaacgaga aggtgctgcc caagcacagc ctgctgtacg agtacttcac cgtgtataac
     2881 gagctgacca aagtgaaata cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
     2941 gagcagaaaa aggccatcgt ggacctgctg ttcaagacca accggaaagt gaccgtgaag
     3001 cagctgaaag aggactactt caagaaaatc gagtgcttcg actccgtgga aatctccggc
     3061 gtggaagatc ggttcaacgc ctccctgggc acataccacg atctgctgaa aattatcaag
     3121 gacaaggact tcctggacaa tgaggaaaac gaggacattc tggaagatat cgtgctgacc
     3181 ctgacactgt ttgaggacag agagatgatc gaggaacggc tgaaaaccta tgcccacctg
     3241 ttcgacgaca aagtgatgaa gcagctgaag cggcggagat acaccggctg gggcaggctg
     3301 agccggaagc tgatcaacgg catccgggac aagcagtccg gcaagacaat cctggatttc
     3361 ctgaagtccg acggcttcgc caacagaaac ttcatgcagc tgatccacga cgacagcctg
     3421 acctttaaag aggacatcca gaaagcccag gtgtccggcc agggcgatag cctgcacgag
     3481 cacattgcca atctggccgg cagccccgcc attaagaagg gcatcctgca gacagtgaag
     3541 gtggtggacg agctcgtgaa agtgatgggc cggcacaagc ccgagaacat cgtgatcgaa
     3601 atggccagag agaaccagac cacccagaag ggacagaaga acagccgcga gagaatgaag
     3661 cggatcgaag agggcatcaa agagctgggc agccagatcc tgaaagaaca ccccgtggaa
     3721 aacacccagc tgcagaacga gaagctgtac ctgtactacc tgcagaatgg gcgggatatg
     3781 tacgtggacc aggaactgga catcaaccgg ctgtccgact acgatgtgga ccatatcgtg
     3841 cctcagagct ttctgaagga cgactccatc gacaacaagg tgctgaccag aagcgacaag
     3901 aaccggggca agagcgacaa cgtgccctcc gaagaggtcg tgaagaagat gaagaactac
     3961 tggcggcagc tgctgaacgc caagctgatt acccagagaa agttcgacaa tctgaccaag
     4021 gccgagagag gcggcctgag cgaactggat aaggccggct tcatcaagag acagctggtg
     4081 gaaacccggc agatcacaaa gcacgtggca cagatcctgg actcccggat gaacactaag
     4141 tacgacgaga atgacaagct gatccgggaa gtgaaagtga tcaccctgaa gtccaagctg
     4201 gtgtccgatt tccggaagga tttccagttt tacaaagtgc gcgagatcaa caactaccac
     4261 cacgcccacg acgcctacct gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct
     4321 aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
     4381 gccaagagcg agcaggaaat cggcaaggct accgccaagt acttcttcta cagcaacatc
     4441 atgaactttt tcaagaccga gattaccctg gccaacggcg agatccggaa gcggcctctg
     4501 atcgagacaa acggcgaaac cggggagatc gtgtgggata agggccggga ttttgccacc
     4561 gtgcggaaag tgctgagcat gccccaagtg aatatcgtga aaaagaccga ggtgcagaca
     4621 ggcggcttca gcaaagagtc tatcctgccc aagaggaaca gcgataagct gatcgccaga
     4681 aagaaggact gggaccctaa gaagtacggc ggcttcgaca gccccaccgt ggcctattct
     4741 gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
     4801 ctgctgggga tcaccatcat ggaaagaagc agcttcgaga agaatcccat cgactttctg
     4861 gaagccaagg gctacaaaga agtgaaaaag gacctgatca tcaagctgcc taagtactcc
     4921 ctgttcgagc tggaaaacgg ccggaagaga atgctggcct ctgccggcga actgcagaag
     4981 ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc tgtacctggc cagccactat
     5041 gagaagctga agggctcccc cgaggataat gagcagaaac agctgtttgt ggaacagcac
     5101 aagcactacc tggacgagat catcgagcag atcagcgagt tctccaagag agtgatcctg
     5161 gccgacgcta atctggacaa agtgctgtcc gcctacaaca agcaccggga taagcccatc
     5221 agagagcagg ccgagaatat catccacctg tttaccctga ccaatctggg agcccctgcc
     5281 gccttcaagt actttgacac caccatcgac cggaagaggt acaccagcac caaagaggtg
     5341 ctggacgcca ccctgatcca ccagagcatc accggcctgt acgagacacg gatcgacctg
     5401 tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag
     5461 aaaaagtaag aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     5521 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     5581 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     5641 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     5701 ctggggagcg gccgctagtt attaatagta atcaattacg gggtcattag ttcatagccc
     5761 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     5821 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     5881 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     5941 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     6001 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     6061 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     6121 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     6181 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     6241 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     6301 gatccgctag catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     6361 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     6421 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     6481 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     6541 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     6601 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     6661 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     6721 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     6781 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     6841 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     6901 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     6961 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     7021 tgtacaagta aggatccggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca
     7081 ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca
     7141 ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct
     7201 gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat
     7261 aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag
     7321 atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag
     7381 tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt
     7441 tccacataca cttcattctc agtattgttt tgccaagttc taattccatc agaagctggt
     7501 cgacctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     7561 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     7621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     7681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     7741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     7801 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     7861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     7921 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     7981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     8041 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     8101 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     8161 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     8221 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     8281 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     8341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     8401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     8461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     8521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     8581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     8641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     8701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     8761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     8821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     8881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     8941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     9001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     9061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     9121 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     9181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     9241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     9301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     9361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     9421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     9481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     9541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     9601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     9661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     9721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     9781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     9841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     9901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     9961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
    10021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
    10081 gccttttgct ggccttttgc tcacatgtga gggcc
//
