LOCUS       sequence-274783-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized SpCas9-VRER and blasticidin 
            resistance: EFS promoter-VRER-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: VRER variant
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    181..298
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        391..602
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      621..630
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(627..4028,4029..4031,4032..4277,4278..4280,4281..4628,
                     4629..4631,4632..4634,4635..4637,4638..4730)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/G1218R/R1335E/T1337R mutations"
                     /label=Cas9 VRER
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             627..4730
                     /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
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4731..4778
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4779..4802
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4812..4868
                     /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             4869..5267
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5292..5880
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5345..5365)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5763..5774)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5883..5899)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5884..5900)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6405..6585
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6611..6628)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6617..6841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6887..7315
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6974..6993)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7184..7205
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7324..7344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7489..7623
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7550..7569
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7685..7732
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7751..8125
                     /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    8255..8376
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8292..8311)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8346..8365
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8425..8441)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8425..8441)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8438..8460)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8449..8465
                     /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(8473..8479,8480..8497,8498..8503))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8518..8539
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8656..8673)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8827..9415)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8907..8926)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9586..10377,10378..10446))
                     /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     10209..10228
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10447..10551)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10626..10645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10817..11196
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11198..11396
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11351..11371
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11414..11594
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11641..11766
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12259..12492
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12677..12721
                     /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 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
       61 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
      121 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa
      181 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
      241 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
      301 ggtttattac agggacagca gagatccagt ttggttaatt agctagctag gtcttgaaag
      361 gagtgggaat tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
      421 agaagttggg gggaggggtc ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa
      481 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
      541 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
      601 aggaccggtt ctagagcgct gccaccatgg acaagaagta cagcatcggc ctggacatcg
      661 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
      721 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
      781 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
      841 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
      901 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
      961 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     1021 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     1081 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     1141 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     1201 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     1261 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     1321 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     1381 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     1441 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     1501 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     1561 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     1621 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     1681 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     1741 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     1801 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     1861 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     1921 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     1981 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     2041 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     2101 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     2161 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     2221 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     2281 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     2341 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     2401 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     2461 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     2521 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     2581 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     2641 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     2701 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     2761 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     2821 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     2881 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     2941 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     3001 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     3061 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     3121 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     3181 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     3241 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     3301 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     3361 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     3421 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     3481 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     3541 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     3601 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     3661 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     3721 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     3781 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     3841 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     3901 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     3961 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     4021 gcggcttcgt gagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     4081 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     4141 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     4201 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     4261 gaatgctggc ctctgcccgg gaactgcaga agggaaacga actggccctg ccctccaaat
     4321 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     4381 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     4441 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     4501 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     4561 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     4621 accggaagga gtaccggagc accaaagagg tgctggacgc caccctgatc caccagagca
     4681 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aagcgacctg
     4741 ccgccacaaa gaaggctgga caggctaaga agaagaaaga ttacaaagac gatgacgata
     4801 agggatccgg cgcaacaaac ttctctctgc tgaaacaagc cggagatgtc gaagagaatc
     4861 ctggaccgat ggccaagcct ttgtctcaag aagaatccac cctcattgaa agagcaacgg
     4921 ctacaatcaa cagcatcccc atctctgaag actacagcgt cgccagcgca gctctctcta
     4981 gcgacggccg catcttcact ggtgtcaatg tatatcattt tactggggga ccttgtgcag
     5041 aactcgtggt gctgggcact gctgctgctg cggcagctgg caacctgact tgtatcgtcg
     5101 cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg acggtgccga caggtgcttc
     5161 tcgatctgca tcctgggatc aaagccatag tgaaggacag tgatggacag ccgacggcag
     5221 ttgggattcg tgaattgctg ccctctggtt atgtgtggga gggctaagaa ttcgatatca
     5281 agcttatcgg taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     5341 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     5401 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     5461 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     5521 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     5581 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     5641 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     5701 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     5761 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     5821 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     5881 atcgataccg tcgacctcga gacctagaaa aacatggagc aatcacaagt agcaatacag
     5941 cagctaccaa tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc
     6001 cagtcacacc tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     6061 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata
     6121 tccttgatct gtggatctac cacacacaag gctacttccc tgattggcag aactacacac
     6181 cagggccagg gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg
     6241 agcaagagaa ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga
     6301 gcctgcatgg gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc
     6361 tagcatttca tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag
     6421 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     6481 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     6541 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc cgtttaaacc
     6601 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     6661 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     6721 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     6781 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     6841 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     6901 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6961 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     7021 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     7081 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     7141 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     7201 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     7261 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     7321 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     7381 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     7441 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     7501 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     7561 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     7621 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
     7681 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     7741 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
     7801 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
     7861 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
     7921 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
     7981 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
     8041 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
     8101 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
     8161 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     8221 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     8281 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     8341 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     8401 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     8461 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     8521 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     8581 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     8641 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     8701 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     8761 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     8821 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     8881 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     8941 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     9001 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     9061 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     9121 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     9181 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     9241 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     9301 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     9361 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     9421 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     9481 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     9541 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     9601 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     9661 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     9721 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     9781 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     9841 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     9901 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     9961 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
    10021 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
    10081 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
    10141 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
    10201 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
    10261 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
    10321 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
    10381 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
    10441 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
    10501 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
    10561 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
    10621 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
    10681 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
    10741 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
    10801 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
    10861 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
    10921 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
    10981 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
    11041 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
    11101 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
    11161 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
    11221 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
    11281 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
    11341 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg
    11401 ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
    11461 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
    11521 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
    11581 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
    11641 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
    11701 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
    11761 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
    11821 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
    11881 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
    11941 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
    12001 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
    12061 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
    12121 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
    12181 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
    12241 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
    12301 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
    12361 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
    12421 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
    12481 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
    12541 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
    12601 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
    12661 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
    12721 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
    12781 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagtt
//
