LOCUS       Exported                9288 bp DNA     circular SYN 27-FEB-2025
DEFINITION  Cas9 from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pSpCas9(BB)-2A-GFP (PX458)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9288)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 9288)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/nprot.2013.143"; journalName: "Nat Protoc"; date: 
            "2013-11"; volume: "8"; issue: "11"; pages: "2281-308"
FEATURES             Location/Qualifiers
     source          1..9288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             827..874
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             890..943
                     /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             944..1657
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             944..1657
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1247..1266)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1594..1615
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1685..1702)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1691..1898
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   1907..2047
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1907..2036
                     /label=AAV2 ITR
     rep_origin      2122..2577
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2209..2228)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2419..2440
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2594..2613)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2713..2735
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2773..2791)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2859..2963
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2964..3824
                     /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(3182..3201)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3995..4583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4484..4503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4645..4893
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4645..4665
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4816..4835
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4912..4987
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        5084..5369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        5371..5648
                     /label=chicken beta-actin promoter
     intron          5649..5876
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      5888..5897
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5897..5962
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5969..5989
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6014..9288,1..826)
                     /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             join(6014..9288,1..826)
                     /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"
ORIGIN
        1 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa
       61 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
      121 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
      181 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
      241 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
      301 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
      361 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
      421 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
      481 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
      541 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
      601 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
      661 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg
      721 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
      781 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc
      841 cacgaaaaag gccggccagg caaaaaagaa aaaggaattc ggcagtggag agggcagagg
      901 aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccagtgagca agggcgagga
      961 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1021 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1081 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1141 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1201 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1261 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1321 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1381 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1441 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1501 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     1561 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1621 cgccgggatc actctcggca tggacgagct gtacaaggaa ttctaactag agctcgctga
     1681 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1741 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1801 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1861 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg
     1921 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     1981 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     2041 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2101 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2161 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     2221 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2281 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     2341 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2401 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     2461 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     2521 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     2581 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     2641 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     2701 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     2761 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2821 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2881 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2941 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3001 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3061 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3121 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3181 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3241 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3301 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3361 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3421 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3481 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3541 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3601 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3661 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     3721 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3781 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3841 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3901 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3961 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4021 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4081 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4141 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4201 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4321 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4381 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4441 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4501 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4561 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4621 tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc atatttgcat
     4681 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
     4741 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     4801 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     4861 gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc tgttttagag
     4921 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     4981 tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt
     5041 agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgttaca taacttacgg
     5101 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca atagtaacgc
     5161 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     5221 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     5281 ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     5341 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
     5401 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
     5461 gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg
     5521 aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc
     5581 gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc
     5641 ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc
     5701 gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc
     5761 cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga tggttggttg
     5821 gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt tttcaggttg
     5881 gaccggtgcc accatggact ataaggacca cgacggagac tacaaggatc atgatattga
     5941 ttacaaagac gatgacgata agatggcccc aaagaagaag cggaaggtcg gtatccacgg
     6001 agtcccagca gccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg
     6061 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
     6121 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
     6181 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
     6241 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
     6301 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
     6361 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
     6421 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
     6481 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
     6541 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
     6601 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
     6661 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
     6721 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
     6781 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
     6841 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
     6901 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
     6961 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
     7021 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
     7081 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
     7141 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
     7201 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
     7261 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
     7321 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
     7381 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
     7441 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
     7501 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
     7561 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
     7621 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
     7681 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
     7741 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
     7801 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
     7861 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
     7921 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
     7981 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
     8041 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
     8101 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
     8161 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
     8221 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
     8281 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
     8341 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
     8401 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
     8461 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
     8521 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
     8581 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
     8641 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
     8701 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
     8761 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
     8821 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
     8881 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
     8941 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
     9001 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
     9061 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
     9121 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
     9181 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
     9241 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaa
//
