LOCUS       sequence-203308-        9253 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  CRISPR knockout. Expresses Cas9, EGFP, and sgRNA targeting TIAL1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 9253)
  AUTHORS   Meyer C, Garzia A, Mazzola M, Gerstberger S, Molina H, Tuschl T
  TITLE     The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated 
            Stress Response and Cell Cycle Progression.
  JOURNAL   Mol Cell. 2018 Feb 15;69(4):622-635.e6. doi: 
            10.1016/j.molcel.2018.01.011.
  PUBMED    29429924
REFERENCE   2  (bases 1 to 9253)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2018.01"; journalName: "Mol Cell"; date: 
            "2018-02-15- 15"; volume: "69"; issue: "4"; pages: "622-635.e6"
COMMENT     Sequence Label: px458_2A_GFP_sgRNA_TIAL1
FEATURES             Location/Qualifiers
     source          1..9253
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          124..351
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      363..372
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             372..437
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             444..464
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             489..4589
                     /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             489..4589
                     /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             4590..4637
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4653..4706
                     /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(4707..4709,4710..5420)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4707..4709,4710..5420)
                     /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(5010..5029)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5357..5378
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5448..5465)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5454..5661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5670..5810
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5670..5799
                     /label=AAV2 ITR
     rep_origin      5885..6340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5972..5991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6182..6203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6357..6376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6476..6498
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6536..6554)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6622..6726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6727..6795,6796..7587)
                     /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(6945..6964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7758..8346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8247..8266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8408..8656
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8408..8428
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8579..8598
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8678..8753
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8850..9135
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
       61 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      121 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      181 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      241 cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg
      301 gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg
      361 gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca
      421 aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc
      481 cagcagccga caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg
      541 ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg
      601 accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag
      661 ccgaggccac ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga
      721 tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc
      781 acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct
      841 tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga
      901 gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg
      961 cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca
     1021 gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa
     1081 accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga
     1141 gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg
     1201 gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg
     1261 ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc
     1321 tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg
     1381 ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg
     1441 cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg
     1501 tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct
     1561 acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca
     1621 tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc
     1681 tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc
     1741 tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa
     1801 agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa
     1861 acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg
     1921 aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg
     1981 ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca
     2041 ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct
     2101 tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag
     2161 tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg
     2221 aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga
     2281 aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata
     2341 tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct
     2401 atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct
     2461 ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa
     2521 tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg
     2581 acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata
     2641 gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc
     2701 agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca
     2761 tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg
     2821 agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac
     2881 accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg
     2941 ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg
     3001 accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca
     3061 gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga
     3121 tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca
     3181 atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga
     3241 gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga
     3301 tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga
     3361 agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg cgcgagatca
     3421 acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca
     3481 aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc
     3541 ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct
     3601 acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga
     3661 agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg
     3721 attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg
     3781 aggtgcagac aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc
     3841 tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg
     3901 tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga
     3961 gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca
     4021 tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc
     4081 ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg
     4141 aactgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg
     4201 ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg
     4261 tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga
     4321 gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg
     4381 ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg accaatctgg
     4441 gagcccctgc cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca
     4501 ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac
     4561 ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc
     4621 aggcaaaaaa gaaaaaggaa ttcggcagtg gagagggcag aggaagtctg ctaacatgcg
     4681 gtgacgtcga ggagaatcct ggcccagtga gcaagggcga ggagctgttc accggggtgg
     4741 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     4801 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     4861 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     4921 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     4981 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     5041 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     5101 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     5161 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     5221 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     5281 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     5341 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     5401 gcatggacga gctgtacaag gaattctaac tagagctcgc tgatcagcct cgactgtgcc
     5461 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5521 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5581 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5641 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     5701 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     5761 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     5821 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     5881 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5941 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     6001 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6061 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     6121 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     6181 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     6241 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     6301 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     6361 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6421 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6481 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     6541 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     6601 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6661 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6721 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6781 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6841 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6901 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6961 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7021 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7081 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     7141 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     7201 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     7261 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     7321 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     7381 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     7441 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     7501 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     7561 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     7621 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     7681 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7741 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7801 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7861 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7921 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7981 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     8041 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     8101 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     8161 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     8221 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     8281 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     8341 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     8401 cacatgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt
     8461 tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg
     8521 acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga
     8581 ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg
     8641 gaaaggacga aacaccgaaa tcagcatttg cccccttgtt ttagagctag aaatagcaag
     8701 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg
     8761 ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa
     8821 ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct
     8881 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
     8941 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
     9001 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
     9061 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
     9121 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
     9181 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
     9241 gcgggggggg ggg
//
