LOCUS       sequence-274782-       12838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human codon-optimized SpCas9-VQR and blasticidin 
            resistance: EFS promoter-VQR-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: VQR variant
FEATURES             Location/Qualifiers
     source          1..12838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    212..329
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        422..633
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      652..661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(658..4059,4060..4062,4063..4659,4660..4662,4663..4665,
                     4666..4668,4669..4761)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /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
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             658..4761
                     /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
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4762..4809
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4810..4833
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             4843..4899
                     /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             4900..5298
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5323..5911
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5376..5396)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5794..5805)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5914..5930)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5915..5931)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6436..6616
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(6642..6659)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6648..6872
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6918..7346
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7005..7024)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7215..7236
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7355..7375)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      7520..7654
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7581..7600
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7716..7763
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7782..8156
                     /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    8286..8407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8323..8342)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8377..8396
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8456..8472)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8456..8472)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8469..8491)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8480..8496
                     /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(8504..8510,8511..8528,8529..8534))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8549..8570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8687..8704)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8858..9446)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8938..8957)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9617..10408,10409..10477))
                     /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     10240..10259
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10478..10582)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10657..10676)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10848..11227
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11229..11427
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11382..11402
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11445..11625
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11672..11797
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12290..12523
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12708..12752
                     /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 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
       61 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
      121 agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat
      181 tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
      241 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
      301 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
      361 tttggttaat tagctagcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
      421 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
      481 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
      541 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
      601 ctttttcgca acgggtttgc cgccagaaca caggaccggt tctagagcgc tgccaccatg
      661 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc
      721 accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac
      781 agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc
      841 acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat
      901 ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg
      961 gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac
     1021 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa
     1081 ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg
     1141 atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1201 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc
     1261 aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg
     1321 ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg
     1381 attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat
     1441 gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     1501 atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg
     1561 ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg
     1621 atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag
     1681 cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc
     1741 tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa
     1801 aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag
     1861 cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc
     1921 attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag
     1981 aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga
     2041 ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg
     2101 gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac
     2161 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat
     2221 aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc
     2281 ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg
     2341 aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc
     2401 ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc
     2461 aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg
     2521 accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac
     2581 ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg
     2641 ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat
     2701 ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc
     2761 ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac
     2821 gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg
     2881 aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc
     2941 gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg
     3001 aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg
     3061 gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat
     3121 atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc
     3181 gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac
     3241 aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac
     3301 tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc
     3361 aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg
     3421 gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact
     3481 aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag
     3541 ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac
     3601 caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac
     3661 cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg
     3721 atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac
     3781 atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct
     3841 ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc
     3901 accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag
     3961 acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc
     4021 agaaagaagg actgggaccc taagaagtac ggcggcttcg tgagccccac cgtggcctat
     4081 tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa
     4141 gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt
     4201 ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac
     4261 tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag
     4321 aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac
     4381 tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag
     4441 cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc
     4501 ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc
     4561 atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct
     4621 gccgccttca agtactttga caccaccatc gaccggaagc agtaccggag caccaaagag
     4681 gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac
     4741 ctgtctcagc tgggaggcga caagcgacct gccgccacaa agaaggctgg acaggctaag
     4801 aagaagaaag attacaaaga cgatgacgat aagggatccg gcgcaacaaa cttctctctg
     4861 ctgaaacaag ccggagatgt cgaagagaat cctggaccga tggccaagcc tttgtctcaa
     4921 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa
     4981 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     5041 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     5101 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     5161 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     5221 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     5281 tatgtgtggg agggctaaga attcgatatc aagcttatcg gtaatcaacc tctggattac
     5341 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     5401 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     5461 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     5521 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     5581 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     5641 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     5701 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     5761 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     5821 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     5881 agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg agacctagaa
     5941 aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta
     6001 gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca
     6061 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     6121 gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa
     6181 ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc
     6241 tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa
     6301 ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga
     6361 gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg
     6421 catccggact gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     6481 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     6541 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     6601 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
     6661 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     6721 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     6781 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     6841 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     6901 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     6961 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     7021 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     7081 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     7141 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     7201 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     7261 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     7321 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
     7381 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     7441 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     7501 aattagtcag caaccatagt cccgccccta actccgccca gttccgccca ttctccgccc
     7561 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     7621 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     7681 gcttgtatat ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt
     7741 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg
     7801 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     7861 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     7921 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     7981 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     8041 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     8101 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     8161 tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     8221 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     8281 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     8341 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     8401 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     8461 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     8521 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     8581 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     8641 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     8701 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     8761 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     8821 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     8881 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     8941 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     9001 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     9061 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     9121 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     9181 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     9241 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     9301 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     9361 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     9421 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     9481 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     9541 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     9601 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     9661 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     9721 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     9781 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     9841 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     9901 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     9961 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
    10021 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
    10081 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
    10141 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
    10201 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
    10261 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
    10321 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
    10381 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
    10441 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
    10501 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
    10561 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
    10621 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
    10681 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
    10741 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
    10801 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
    10861 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
    10921 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
    10981 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
    11041 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
    11101 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
    11161 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
    11221 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
    11281 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
    11341 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
    11401 tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct ctctggttag
    11461 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
    11521 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
    11581 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag
    11641 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga
    11701 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
    11761 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
    11821 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
    11881 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
    11941 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
    12001 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
    12061 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
    12121 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
    12181 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
    12241 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
    12301 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg
    12361 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
    12421 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
    12481 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
    12541 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
    12601 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca
    12661 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag
    12721 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc
    12781 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagt
//
