LOCUS       sequence-203427-        9115 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  CRISPR knockout. Expresses Cas9, EGFP, and sgRNA targeting EIF2AK2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 9115)
  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 9115)
  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_EIF2AK2
FEATURES             Location/Qualifiers
     source          1..9115
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      227..236
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             236..301
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             308..328
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             353..4453
                     /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             353..4453
                     /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             4454..4501
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4517..4570
                     /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(4571..4573,4574..5284)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4571..4573,4574..5284)
                     /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(4874..4893)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5221..5242
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5312..5329)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5318..5525
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5534..5674
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5534..5663
                     /label=AAV2 ITR
     rep_origin      5749..6204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5836..5855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6046..6067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6221..6240)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6340..6362
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6400..6418)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6486..6590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6591..6659,6660..7451)
                     /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(6809..6828)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7622..8210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8111..8130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8272..8519
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8272..8292
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8443..8462
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8541..8616
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8713..8998
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac
       61 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt
      121 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt
      181 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta
      241 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
      301 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
      361 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
      421 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
      481 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
      541 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
      601 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
      661 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
      721 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
      781 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
      841 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
      901 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
      961 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     1021 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct
     1081 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     1141 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     1201 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     1261 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     1321 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     1381 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     1441 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     1501 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     1561 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     1621 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     1681 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     1741 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     1801 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     1861 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     1921 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     1981 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     2041 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     2101 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     2161 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     2221 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     2281 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     2341 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     2401 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     2461 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     2521 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     2581 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     2641 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     2701 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     2761 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     2821 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca
     2881 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     2941 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     3001 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     3061 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     3121 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     3181 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     3241 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     3301 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     3361 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     3421 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     3481 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     3541 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     3601 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     3661 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     3721 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct
     3781 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     3841 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     3901 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     3961 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     4021 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     4081 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     4141 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     4201 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     4261 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     4321 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     4381 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     4441 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     4501 ggaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa
     4561 tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     4621 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     4681 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     4741 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     4801 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     4861 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     4921 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     4981 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     5041 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     5101 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     5161 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     5221 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     5281 caaggaattc taactagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     5341 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5401 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     5461 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct
     5521 ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     5581 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     5641 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     5701 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     5761 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     5821 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     5881 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     5941 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     6001 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     6061 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     6121 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     6181 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     6241 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     6301 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     6361 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     6421 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     6481 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     6541 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     6601 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     6661 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     6721 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     6781 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     6841 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     6901 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     6961 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     7021 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     7081 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     7141 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     7201 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     7261 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     7321 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     7381 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     7441 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     7501 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     7561 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     7621 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     7681 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     7741 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     7801 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     7861 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     7921 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     7981 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     8041 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     8101 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     8161 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     8221 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tgagggccta
     8281 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
     8341 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     8401 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     8461 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacg
     8521 taatacatac cgtcagaagc gttttagagc tagaaatagc aagttaaaat aaggctagtc
     8581 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga gctagaaata
     8641 gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca gacaaatggc
     8701 tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     8761 cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt
     8821 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     8881 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac
     8941 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     9001 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     9061 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg ggggg
//
