LOCUS       sequence-343189-        9699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9-2A-GFP expression vector bearing two (one additional) 
            independent sgRNA cassettes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9699)
  AUTHORS   Wu HJ, Landshammer A, Stamenova EK, Bolondi A, Kretzmer H, Meissner 
            A, Michor F
  TITLE     Topological isolation of developmental regulators in mammalian 
            genomes.
  JOURNAL   Nat Commun. 2021 Aug 12;12(1):4897. doi: 10.1038/s41467-021-24951-7.
  PUBMED    34385432
REFERENCE   2  (bases 1 to 9699)
  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"; journalName: "Nat 
            Commun."; date: "2021-08-12"; volume: "12(1):4897. doi"; pages: " 
            10.1038/s41467-021-24951-7"
COMMENT     Sequence Label: 2X_pX458_pSpCas9(BB)-2A-GFP
FEATURES             Location/Qualifiers
     source          1..9699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        707..782
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        851..1136
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1416..1643
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1655..1664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1664..1729
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1736..1756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1781..5881
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1781..5881
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5882..5929
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5945..5998
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(5999..6001,6002..6712)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5999..6001,6002..6712)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6302..6321)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6649..6670
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6740..6757)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6746..6953
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6962..7102
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6962..7091
                     /label=AAV2 ITR
     rep_origin      7177..7632
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7264..7283)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7474..7495
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7649..7668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7768..7790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7828..7846)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7914..8018
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8019..8087,8088..8879)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(8237..8256)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9050..9638
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9539..9558
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtacca atatgcattt tcccatgatt ccttcatatt tgcatatacg
      481 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      541 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      601 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      661 atatatcttg tggaaaggac gaaacaccgg aagagcgagc tcttctgttt tagagctaga
      721 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      781 gcttttttgt tttcctgcag gagatttagc gcgtgcgcca attctgcaga caaatggctc
      841 tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      901 cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      961 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     1021 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct
     1081 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg
     1141 aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
     1201 tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc
     1261 gcgcgccggg gggggggggg gggggggggg gggggggsgg ggsgrggcgg rgaggkgcgg
     1321 cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc
     1381 ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc
     1441 ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac
     1501 tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga
     1561 ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc
     1621 tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata aggaccacga
     1681 cggagactac aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa
     1741 gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg
     1801 cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc
     1861 cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat
     1921 cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc
     1981 cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa
     2041 cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga
     2101 agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta
     2161 ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa
     2221 ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt
     2281 cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct
     2341 ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc
     2401 caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca
     2461 gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct
     2521 gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa
     2581 ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga
     2641 cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt
     2701 gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca
     2761 ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa
     2821 agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag
     2881 ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga
     2941 actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg
     3001 cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga
     3061 tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat
     3121 cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa
     3181 gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc
     3241 ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct
     3301 gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa
     3361 atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat
     3421 cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta
     3481 cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa
     3541 cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga
     3601 caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga
     3661 cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat
     3721 gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa
     3781 cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt
     3841 cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat
     3901 ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc
     3961 cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt
     4021 gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca
     4081 gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat
     4141 caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa
     4201 cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact
     4261 ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa
     4321 ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga
     4381 caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa
     4441 cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct
     4501 gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac
     4561 aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa
     4621 gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa
     4681 ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta
     4741 cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt
     4801 cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga
     4861 aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac
     4921 cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga
     4981 aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag
     5041 catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga
     5101 gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc
     5161 taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa
     5221 agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat
     5281 catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa
     5341 agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa
     5401 cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct
     5461 gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc
     5521 ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga
     5581 gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga
     5641 caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa
     5701 tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga
     5761 caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat
     5821 ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga
     5881 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg aattcggcag
     5941 tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagt
     6001 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     6061 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     6121 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     6181 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     6241 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     6301 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     6361 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     6421 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     6481 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     6541 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     6601 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     6661 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca aggaattcta
     6721 actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     6781 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     6841 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     6901 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     6961 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     7021 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     7081 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     7141 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     7201 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     7261 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     7321 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     7381 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     7441 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     7501 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     7561 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     7621 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     7681 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     7741 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     7801 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     7861 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     7921 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     7981 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     8041 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     8101 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     8161 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     8221 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     8281 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     8341 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     8401 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     8461 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     8521 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     8581 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     8641 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     8701 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     8761 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     8821 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     8881 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     8941 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     9001 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     9061 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     9121 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     9181 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     9241 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     9301 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     9361 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     9421 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     9481 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     9541 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     9601 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     9661 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
