LOCUS       sequence-339219-        9262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 with 3' gRNA for paired CRISPR KO of miR-290-295 cluster.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9262)
  AUTHORS   Schaefer M, Nabih A, Spies D, Hermes V, Bodak M, Wischnewski H, 
            Stalder P, Ngondo RP, Liechti LA, Sajic T, Aebersold R, Gatfield D, 
            Ciaudo C
  TITLE     Global and precise identification of functional miRNA targets in 
            mESCs by integrative analysis.
  JOURNAL   EMBO Rep. 2022 Jul 28:e54762. doi: 10.15252/embr.202254762.
  PUBMED    35899551
REFERENCE   2  (bases 1 to 9262)
  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: "EMBO Rep. 
            2022 Jul 28:e54762. doi: 10.15252/embr.202254762."
COMMENT     Sequence Label: pX458_miR-290-295KO-gRNA2
FEATURES             Location/Qualifiers
     source          1..9262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        443..728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          979..1206
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1218..1227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1227..1292
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1299..1319
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1344..5444
                     /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             1344..5444
                     /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             5445..5492
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5508..5561
                     /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(5562..5564,5565..6275)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5562..5564,5565..6275)
                     /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(5865..5884)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6212..6233
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6303..6320)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6309..6516
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6525..6665
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6525..6654
                     /label=AAV2 ITR
     rep_origin      6740..7195
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6827..6846)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7037..7058
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7212..7231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7331..7353
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7391..7409)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7477..7581
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7582..7650,7651..8442)
                     /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(7800..7819)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8613..9201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9102..9121
                     /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 tggttgctcc catagcacct gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca
      421 gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac
      481 cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac
      541 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      601 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc
      661 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      721 ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc
      781 cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg
      841 gggggggggg gggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      901 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      961 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
     1021 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
     1081 cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg gtttaaggga
     1141 tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga aatcactttt
     1201 tttcaggttg gaccggtgcc accatggact ataaggacca cgacggagac tacaaggatc
     1261 atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag cggaaggtcg
     1321 gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac atcggcacca
     1381 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     1441 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     1501 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     1561 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     1621 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     1681 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     1741 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     1801 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     1861 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     1921 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     1981 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     2041 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     2101 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     2161 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     2221 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     2281 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     2341 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     2401 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     2461 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     2521 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     2581 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     2641 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     2701 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     2761 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     2821 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     2881 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     2941 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     3001 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     3061 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     3121 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     3181 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     3241 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     3301 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     3361 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     3421 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     3481 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     3541 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     3601 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     3661 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     3721 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     3781 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     3841 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     3901 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     3961 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     4021 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     4081 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     4141 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     4201 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     4261 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     4321 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     4381 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     4441 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     4501 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     4561 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     4621 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     4681 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     4741 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     4801 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     4861 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     4921 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     4981 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     5041 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     5101 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     5161 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     5221 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     5281 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     5341 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     5401 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg ccggcggcca
     5461 cgaaaaaggc cggccaggca aaaaagaaaa aggaattcgg cagtggagag ggcagaggaa
     5521 gtctgctaac atgcggtgac gtcgaggaga atcctggccc agtgagcaag ggcgaggagc
     5581 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     5641 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     5701 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     5761 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     5821 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     5881 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     5941 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     6001 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     6061 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     6121 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     6181 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     6241 ccgggatcac tctcggcatg gacgagctgt acaaggaatt ctaactagag ctcgctgatc
     6301 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6361 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6421 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     6481 ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat
     6541 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     6601 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     6661 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     6721 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     6781 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     6841 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     6901 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     6961 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     7021 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     7081 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     7141 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     7201 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     7261 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     7321 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     7381 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     7441 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     7501 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     7561 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     7621 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     7681 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     7741 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     7801 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7861 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7921 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     7981 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     8041 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     8101 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     8161 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     8221 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     8281 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     8341 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     8401 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     8461 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     8521 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     8581 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     8641 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     8701 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     8761 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     8821 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     8881 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     8941 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     9001 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     9061 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     9121 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     9181 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     9241 tgctggcctt ttgctcacat gt
//
