LOCUS       sequence-274351-        9289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA for CRISPR/Cas9 knockout of human TRIM21.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9289)
  AUTHORS   Park JS, Burckhardt CJ, Lazcano R, Solis LM, Isogai T, Li L, Chen 
            CS, Gao B, Minna JD, Bachoo R, DeBerardinis RJ, Danuser G
  TITLE     Mechanical regulation of glycolysis via cytoskeleton architecture.
  JOURNAL   Nature. 2020 Feb 12. pii: 10.1038/s41586-020-1998-1. doi: 
            10.1038/s41586-020-1998-1.
  PUBMED    32051585
REFERENCE   2  (bases 1 to 9289)
  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: "Nature. 
            2020 Feb 12. pii: 10.1038/s41586-020-1998-1. doi: 
            10.1038/s41586-020-1998-1."
COMMENT     Sequence Label: pSpCAS9 wt (BB)-2A-GFP targeting human TRIM21
FEATURES             Location/Qualifiers
     source          1..9289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          220..447
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      459..468
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             468..533
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             540..560
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             585..4685
                     /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             585..4685
                     /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             4686..4733
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4749..4802
                     /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(4803..4805,4806..5516)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4803..4805,4806..5516)
                     /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(5106..5125)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5453..5474
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5544..5561)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5550..5757
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5766..5906
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5766..5895
                     /label=AAV2 ITR
     rep_origin      5981..6436
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6068..6087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6278..6299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6453..6472)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6572..6594
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6632..6650)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6718..6822
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6823..6891,6892..7683)
                     /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(7041..7060)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7854..8442
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8343..8362
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8504..8752
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8504..8524
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8675..8694
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8773..8848
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8945..9230
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        join(9232..9289,1..219)
                     /label=chicken beta-actin promoter
ORIGIN
        1 aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg
       61 gggcgcgcgc crggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
      121 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      181 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      241 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      301 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagctgagc
      361 aagaggtaag ggtttaaggg atggttggtt ggtggggtat taatgtttaa ttacctggag
      421 cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc caccatggac tataaggacc
      481 acgacggaga ctacaaggat catgatattg attacaaaga cgatgacgat aagatggccc
      541 caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaag aagtacagca
      601 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
      661 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
      721 tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa
      781 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
      841 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
      901 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
      961 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     1021 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     1081 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     1141 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     1201 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     1261 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     1321 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     1381 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     1441 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     1501 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     1561 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     1621 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     1681 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     1741 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     1801 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     1861 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     1921 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     1981 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     2041 ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg
     2101 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     2161 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     2221 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     2281 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     2341 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     2401 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     2461 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     2521 tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga
     2581 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     2641 gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc tttaaagagg
     2701 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     2761 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     2821 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     2881 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     2941 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     3001 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     3061 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     3121 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     3181 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     3241 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     3301 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga
     3361 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     3421 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     3481 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     3541 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     3601 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     3661 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     3721 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     3781 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     3841 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     3901 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     3961 accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     4021 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     4081 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     4141 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     4201 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     4261 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     4321 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     4381 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     4441 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     4501 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     4561 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     4621 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     4681 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aaggaattcg
     4741 gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc
     4801 cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4861 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     4921 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     4981 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     5041 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     5101 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     5161 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     5221 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     5281 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     5341 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     5401 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     5461 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat
     5521 tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     5581 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5641 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5701 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg
     5761 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     5821 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     5881 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     5941 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     6001 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     6061 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     6121 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     6181 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     6241 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     6301 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     6361 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     6421 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     6481 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     6541 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     6601 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     6661 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     6721 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     6781 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6841 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6901 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6961 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     7021 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     7081 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     7141 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     7201 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     7261 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     7321 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     7381 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     7441 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     7501 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     7561 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     7621 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     7681 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     7741 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     7801 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7861 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7921 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7981 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     8041 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     8101 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     8161 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     8221 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     8281 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     8341 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8401 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8461 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat
     8521 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     8581 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     8641 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     8701 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaaacacc
     8761 gtgaccacgc cagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca
     8821 acttgaaaaa gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta
     8881 aaataaggct agtccgtttt tagcgcgtgc gccaattctg cagacaaatg gctctagagg
     8941 tacccgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     9001 cattgacgtc aatagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     9061 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     9121 ttgacgtcaa tgacggtaaa tggcccgcct ggcattgtgc ccagtacatg accttatggg
     9181 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga
     9241 gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccc
//
