LOCUS       sequence-274767-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized hyper-accurate SpCas9 and 
            blasticidin resistance: EFS promoter-HypaCas9-NLS-FLAG-P2A-BSD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12838)
  AUTHORS   Kim N, Kim HK, Lee S, Seo JH, Choi JW, Park J, Min S, Yoon S, Cho 
            SR, Kim HH
  TITLE     Prediction of the sequence-specific cleavage activity of Cas9 
            variants.
  JOURNAL   Nat Biotechnol. 2020 Nov;38(11):1328-1336. doi: 
            10.1038/s41587-020-0537-9. Epub 2020 Jun 8.
  PUBMED    32514125
REFERENCE   2  (bases 1 to 12838)
  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"; doi: 
            "10.1038/s41587-020-0537-9"; journalName: "Nat Biotechnol"; date: 
            "2020-11"; volume: "38"; issue: "11"; pages: "1328-1336"
COMMENT     Sequence Label: HypaCas9
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    120..237
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        330..541
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      560..569
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(566..2638,2639..2641,2642..2644,2645..2650,2651..2656,
                     2657..2659,2660..4669)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity without compromising on-target activity (Chen 
                     et al., 2017)"
                     /label=HypaCas9
                     /note="carries the mutations N692A, M694A, Q695A, and 
                     H698A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             566..4669
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFA
                     ALIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4670..4717
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4718..4741
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4751..4807
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4808..5206
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5231..5819
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5284..5304)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5702..5713)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5822..5838)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5823..5839)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6344..6524
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6550..6567)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6556..6780
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6826..7254
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6913..6932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7123..7144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7263..7283)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7428..7562
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7489..7508
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7624..7671
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7690..8064
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    8194..8315
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8231..8250)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8285..8304
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8364..8380)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8364..8380)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8377..8399)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8388..8404
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(8412..8418,8419..8436,8437..8442))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8457..8478
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8595..8612)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8766..9354)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8846..8865)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9525..10316,10317..10385))
                     /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     10148..10167
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10386..10490)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10565..10584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10756..11135
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11137..11335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11290..11310
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11353..11533
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11580..11705
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12198..12431
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12616..12660
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
       61 tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat
      121 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
      181 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
      241 gtttattaca gggacagcag agatccagtt tggttaatta gctagctagg tcttgaaagg
      301 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
      361 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
      421 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      481 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
      541 ggaccggttc tagagcgctg ccaccatgga caagaagtac agcatcggcc tggacatcgg
      601 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
      661 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
      721 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
      781 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
      841 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
      901 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
      961 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1021 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1081 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1141 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1201 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     1261 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     1321 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     1381 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     1441 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     1501 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     1561 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     1621 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     1681 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     1741 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     1801 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     1861 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     1921 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     1981 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2041 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2101 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2161 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     2221 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     2281 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     2341 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     2401 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     2461 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     2521 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     2581 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagagc
     2641 cttcgccgcc ctgatcgccg acgacagcct gacctttaaa gaggacatcc agaaagccca
     2701 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     2761 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     2821 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     2881 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     2941 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     3001 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3061 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     3121 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     3181 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     3241 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     3301 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     3361 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     3421 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     3481 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     3541 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     3601 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     3661 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     3721 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     3781 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     3841 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     3901 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     3961 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     4021 caagtccaag aaactgaaga gtgtgaaaga gctactgggg atcaccatca tggaaagaag
     4081 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     4141 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     4201 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     4261 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     4321 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     4381 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     4441 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     4501 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     4561 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     4621 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca agcgacctgc
     4681 cgccacaaag aaggctggac aggctaagaa gaagaaagat tacaaagacg atgacgataa
     4741 gggatccggc gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc
     4801 tggaccgatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     4861 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     4921 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     4981 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     5041 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     5101 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     5161 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagaat tcgatatcaa
     5221 gcttatcggt aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
     5281 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
     5341 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
     5401 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
     5461 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
     5521 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
     5581 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
     5641 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
     5701 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
     5761 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca
     5821 tcgataccgt cgacctcgag acctagaaaa acatggagca atcacaagta gcaatacagc
     5881 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
     5941 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     6001 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     6061 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
     6121 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     6181 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
     6241 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
     6301 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
     6361 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6421 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6481 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagggccc gtttaaaccc
     6541 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     6601 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     6661 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     6721 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     6781 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     6841 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6901 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6961 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     7021 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     7081 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     7141 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     7201 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     7261 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     7321 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     7381 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     7441 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     7501 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     7561 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     7621 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     7681 aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc
     7741 ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac
     7801 ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg
     7861 gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag
     7921 tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc
     7981 ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac
     8041 ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc
     8101 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     8161 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     8221 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     8281 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     8341 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     8401 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     8461 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     8521 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     8581 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     8641 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     8701 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     8761 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     8821 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     8881 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     8941 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     9001 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     9061 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     9121 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     9181 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     9241 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     9301 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     9361 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     9421 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     9481 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     9541 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     9601 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     9661 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     9721 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     9781 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     9841 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     9901 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     9961 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
    10021 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
    10081 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
    10141 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
    10201 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
    10261 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
    10321 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
    10381 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
    10441 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
    10501 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
    10561 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
    10621 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
    10681 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
    10741 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
    10801 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
    10861 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
    10921 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
    10981 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
    11041 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
    11101 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
    11161 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
    11221 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
    11281 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagcgcgt
    11341 tttgcctgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
    11401 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
    11461 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
    11521 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
    11581 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
    11641 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
    11701 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
    11761 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
    11821 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
    11881 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
    11941 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
    12001 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
    12061 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
    12121 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
    12181 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
    12241 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
    12301 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
    12361 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
    12421 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
    12481 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
    12541 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
    12601 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
    12661 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
    12721 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
    12781 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgaca
//
