LOCUS       sequence-200900-        9292 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR knockout. Expresses Cas9, EGFP, and sgRNA targeting TIA1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9292)
  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 9292)
  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_TIA1
FEATURES             Location/Qualifiers
     source          1..9292
                     /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"
     promoter        730..1007
                     /label=chicken beta-actin promoter
     intron          1008..1236
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1248..1257
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1257..1322
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1329..1349
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1374..5474
                     /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             1374..5474
                     /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             5475..5522
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5538..5591
                     /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(5592..5594,5595..6305)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(5592..5594,5595..6305)
                     /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(5895..5914)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6242..6263
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6333..6350)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6339..6546
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6555..6695
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6555..6684
                     /label=AAV2 ITR
     rep_origin      6770..7225
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6857..6876)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7067..7088
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7242..7261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7361..7383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7421..7439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7507..7611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7612..7680,7681..8472)
                     /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(7830..7849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8643..9231
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9132..9151
                     /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 agtttttaca aggtccaatc 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 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc
      901 ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga
      961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcga
     1021 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
     1081 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
     1141 tagctgagca agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat
     1201 tacctggagc acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact
     1261 ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata
     1321 agatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga
     1381 agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg
     1441 agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca
     1501 agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc
     1561 tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag
     1621 agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt
     1681 ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg
     1741 acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg
     1801 acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt
     1861 tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc
     1921 tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca
     1981 gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa
     2041 atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc
     2101 tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac
     2161 tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg
     2221 accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg
     2281 acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga
     2341 gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc
     2401 ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg
     2461 acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg
     2521 acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga
     2581 ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc
     2641 ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc
     2701 tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct
     2761 ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca
     2821 agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca
     2881 acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc
     2941 tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc
     3001 agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc
     3061 tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg
     3121 aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca
     3181 aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga
     3241 cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg
     3301 acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc
     3361 ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga
     3421 agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct
     3481 ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca
     3541 ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg
     3601 tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg
     3661 ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga
     3721 tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca
     3781 cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg
     3841 tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc
     3901 agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc
     3961 ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc
     4021 ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg
     4081 agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa
     4141 cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg
     4201 acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt
     4261 ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg
     4321 cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc
     4381 tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca
     4441 agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga
     4501 actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg
     4561 agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc
     4621 ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg
     4681 gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga
     4741 aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc
     4801 tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc
     4861 tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag
     4921 ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt
     4981 tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa
     5041 acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga
     5101 agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc
     5161 actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg
     5221 acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag
     5281 agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct
     5341 tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg
     5401 acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc
     5461 agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     5521 aggaattcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga
     5581 atcctggccc agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     5641 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     5701 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     5761 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     5821 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     5881 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     5941 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     6001 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
     6061 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
     6121 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     6181 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     6241 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     6301 acaaggaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     6361 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     6421 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     6481 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc
     6541 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     6601 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     6661 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     6721 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     6781 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     6841 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     6901 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     6961 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     7021 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     7081 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg
     7141 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     7201 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     7261 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     7321 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     7381 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     7441 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     7501 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     7561 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     7621 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     7681 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     7741 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     7801 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     7861 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     7921 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7981 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     8041 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     8101 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     8161 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     8221 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     8281 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     8341 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     8401 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     8461 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     8521 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     8581 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     8641 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     8701 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     8761 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     8821 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     8881 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8941 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     9001 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     9061 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     9121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     9181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     9241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
