LOCUS       sequence-339221-        9283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 with 5' 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 9283)
  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 9283)
  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-gRNA3
FEATURES             Location/Qualifiers
     source          1..9283
                     /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          1000..1227
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1239..1248
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1248..1313
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1320..1340
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1365..5465
                     /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             1365..5465
                     /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             5466..5513
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5529..5582
                     /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(5583..5585,5586..6296)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5583..5585,5586..6296)
                     /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(5886..5905)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6233..6254
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6324..6341)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6330..6537
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6546..6686
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6546..6675
                     /label=AAV2 ITR
     rep_origin      6761..7216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6848..6867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7058..7079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7233..7252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7352..7374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7412..7430)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7498..7602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7603..7671,7672..8463)
                     /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(7821..7840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8634..9222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9123..9142
                     /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 ggtctacata atttcaaggg 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 ggggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
      901 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      961 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
     1021 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
     1081 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc
     1141 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag
     1201 cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac tataaggacc
     1261 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
     1321 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
     1381 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
     1441 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
     1501 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
     1561 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     1621 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     1681 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     1741 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     1801 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     1861 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     1921 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     1981 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     2041 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     2101 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     2161 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     2221 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     2281 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     2341 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     2401 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     2461 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     2521 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     2581 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     2641 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     2701 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     2761 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     2821 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     2881 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     2941 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     3001 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     3061 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     3121 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     3181 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     3241 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     3301 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     3361 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     3421 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     3481 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     3541 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     3601 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     3661 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     3721 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     3781 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     3841 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     3901 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     3961 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     4021 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     4081 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     4141 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     4201 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     4261 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     4321 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     4381 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     4441 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     4501 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     4561 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     4621 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     4681 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     4741 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     4801 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     4861 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     4921 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     4981 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     5041 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     5101 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     5161 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     5221 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     5281 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     5341 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     5401 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     5461 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg
     5521 gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     5581 cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     5641 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     5701 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     5761 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     5821 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     5881 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     5941 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     6001 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     6061 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     6121 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     6181 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     6241 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat
     6301 tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     6361 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     6421 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     6481 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     6541 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     6601 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     6661 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     6721 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     6781 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     6841 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     6901 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6961 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     7021 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     7081 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     7141 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     7201 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     7261 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     7321 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     7381 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     7441 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     7501 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     7561 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     7621 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     7681 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     7741 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     7801 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     7861 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     7921 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     7981 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     8041 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     8101 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     8161 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     8221 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     8281 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     8341 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     8401 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     8461 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     8521 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     8581 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     8641 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     8701 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     8761 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     8821 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8881 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8941 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     9001 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     9061 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     9121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     9181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     9241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
