LOCUS       sequence-405306-        9309 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression plasmid of high-fidelity SpCas9 variant 
            SpCas9-HF1 with T2A-EGFP and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9309)
  AUTHORS   Acharya S, Ansari AH, Kumar Das P, Hirano S, Aich M, Rauthan R, 
            Mahato S, Maddileti S, Sarkar S, Kumar M, Phutela R, Gulati S, 
            Rahman A, Goel A, Afzal C, Paul D, Agrawal T, Pulimamidi VK, Jalali 
            S, Nishimasu H, Mariappan I, Nureki O, Maiti S, Chakraborty D
  TITLE     PAM-flexible Engineered FnCas9 variants for robust and ultra-precise
            genome editing and diagnostics.
  JOURNAL   Nat Commun. 2024 Jun 28;15(1):5471. doi: 10.1038/s41467-024-49233-w.
  PUBMED    38942756
REFERENCE   2  (bases 1 to 9309)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-06-28"; volume: "15(1):5471. doi"; pages: " 
            10.1038/s41467-024-49233-w"
COMMENT     Sequence Label: PX458-3xHA-SpCas9-HF1
FEATURES             Location/Qualifiers
     source          1..9309
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1259..1339
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             1346..1366
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1391..2875,2876..2878,2879..3367,3368..3370,3371..3469,
                     3470..3472,3473..4162,4163..4165,4166..5491)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1391..5491
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5492..5539
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5555..5608
                     /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             join(5609..5611,5612..6322)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5609..5611,5612..6322)
                     /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(5912..5931)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6259..6280
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6350..6367)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6356..6563
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6572..6712
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6572..6701
                     /label=AAV2 ITR
     rep_origin      6787..7242
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6874..6893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7084..7105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7259..7278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7378..7400
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7438..7456)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7524..7628
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7629..7697,7698..8489)
                     /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(7847..7866)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8660..9248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9149..9168
                     /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 ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tgggatccta
     1261 cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg cttacccata
     1321 cgatgttcca gattacgcta tggccccaaa gaagaagcgg aaggtcggta tccacggagt
     1381 cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     1441 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     1501 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     1561 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     1621 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     1681 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     1741 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     1801 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     1861 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     1921 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     1981 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     2041 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     2101 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     2161 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     2221 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     2281 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     2341 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     2401 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     2461 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     2521 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     2581 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     2641 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     2701 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     2761 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     2821 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccgcctt
     2881 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     2941 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     3001 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     3061 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     3121 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     3181 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     3241 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     3301 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     3361 ctggggcgcc ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     3421 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgg ccctgatcca
     3481 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     3541 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     3601 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     3661 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     3721 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     3781 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     3841 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     3901 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
     3961 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     4021 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     4081 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     4141 gagacagctg gtggaaaccc gggccatcac aaagcacgtg gcacagatcc tggactcccg
     4201 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     4261 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     4321 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     4381 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     4441 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     4501 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     4561 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     4621 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     4681 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     4741 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     4801 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     4861 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     4921 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     4981 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     5041 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     5101 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     5161 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     5221 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     5281 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     5341 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     5401 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     5461 acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg
     5521 ccaggcaaaa aagaaaaagg aattcggcag tggagagggc agaggaagtc tgctaacatg
     5581 cggtgacgtc gaggagaatc ctggcccagt gagcaagggc gaggagctgt tcaccggggt
     5641 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     5701 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     5761 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     5821 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     5881 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     5941 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     6001 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     6061 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     6121 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     6181 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     6241 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     6301 cggcatggac gagctgtaca aggaattcta actagagctc gctgatcagc ctcgactgtg
     6361 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     6421 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     6481 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     6541 gagaatagca ggcatgctgg ggagcggccg caggaacccc tagtgatgga gttggccact
     6601 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     6661 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     6721 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     6781 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     6841 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     6901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     6961 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     7021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     7081 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     7141 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     7201 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     7261 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     7321 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     7381 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     7441 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     7501 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     7561 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     7621 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     7681 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     7741 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     7801 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     7861 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     7921 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     7981 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     8041 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     8101 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     8161 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     8221 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     8281 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     8341 gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     8401 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     8461 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     8521 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     8581 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     8641 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     8701 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     8761 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     8821 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     8881 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     8941 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     9001 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     9061 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     9121 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     9181 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     9241 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     9301 ctcacatgt
//
