LOCUS Exported 9291 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Knock out of EB3 in human cells by CRISPR/Cas9. ACCESSION . VERSION . KEYWORDS pSpCas9_BB_2A-GFP_MAPRE3-gRNA#2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9291) AUTHORS van Haren J, Charafeddine RA, Ettinger A, Wang H, Hahn KM, Wittmann T TITLE Local control of intracellular microtubule dynamics by EB1 photodissociation. JOURNAL Nat Cell Biol. 2018 Jan 29. pii: 10.1038/s41556-017-0028-5. doi: 10.1038/s41556-017-0028-5. PUBMED 29379139 REFERENCE 2 (bases 1 to 9291) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9291 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 271..346 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 443..728 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" intron 1008..1235 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1247..1256 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1256..1321 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1328..1348 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1373..5473 /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 5474..5521 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5537..5590 /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 5591..6304 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 5591..6304 /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(5633..5654) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5894..5913) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6241..6262 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(6332..6349) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 6338..6545 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6554..6694 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6554..6683 /label=AAV2 ITR rep_origin 6769..7224 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6856..6875) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7066..7087 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(7241..7260) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7360..7382 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7420..7438) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7506..7610 /gene="bla" /label=AmpR promoter CDS 7611..8471 /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(7829..7848) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8642..9230 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9131..9150 /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 tcgccatgat atgcttgcat 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 gcgcgcgccr gggggggggg gggggggggg gggggggggs ggggsgrggc 901 ggrgaggkgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga 961 ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc 1021 gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1081 tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1141 agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1201 acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1261 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1321 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1381 gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1441 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1501 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1561 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1621 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1681 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1741 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1801 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1861 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1921 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1981 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2041 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2101 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2161 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2221 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2281 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2341 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2401 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2461 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2521 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2581 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2641 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2701 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2761 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2821 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2881 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2941 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3001 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3061 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3121 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3181 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3241 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3301 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3361 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3421 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3481 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3541 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3601 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3661 cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3721 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3781 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3841 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3901 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3961 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4021 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4081 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4141 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4201 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4261 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4321 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4381 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4441 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4501 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4561 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4621 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4681 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4741 ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4801 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4861 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4921 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4981 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5041 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5101 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5161 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5221 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5281 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5341 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5401 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5461 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5521 ggaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa 5581 tcctggccca gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 5641 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 5701 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 5761 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 5821 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 5881 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 5941 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 6001 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 6061 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 6121 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 6181 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 6241 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 6301 caaggaattc taactagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca 6361 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 6421 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 6481 gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct 6541 ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 6601 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 6661 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt 6721 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta 6781 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 6841 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 6901 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 6961 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat 7021 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 7081 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc 7141 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 7201 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg 7261 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 7321 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 7381 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 7441 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 7501 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 7561 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 7621 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 7681 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 7741 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 7801 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 7861 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 7921 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 7981 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 8041 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 8101 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 8161 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 8221 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 8281 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca 8341 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 8401 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 8461 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 8521 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 8581 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 8641 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 8701 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 8761 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 8821 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 8881 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 8941 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 9001 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 9061 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 9121 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 9181 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 9241 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg t //