LOCUS Exported 10963 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION For simple determination of the multiplicity of infection (MOI) of a lentiviral CRISPR library by checking EGFP expression (still allowing for puro selection).. ACCESSION . VERSION . KEYWORDS LentiGuide Puro-P2A-EGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10963) AUTHORS Panda SK, Wigerblad G, Jiang L, Jimenez-Andrade Y, Iyer VS, Shen Y, Boddul SV, Guerreiro-Cacais AO, Raposo B, Kasza Z, Wermeling F TITLE IL-4 controls activated neutrophil FcgammaR2b expression and migration into inflamed joints. JOURNAL Proc Natl Acad Sci U S A. 2020 Jan 24. pii: 1914186117. doi: 10.1073/pnas.1914186117. PUBMED 31980518 REFERENCE 2 (bases 1 to 10963) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10963 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 87..1265 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 318..1256 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1213..1233 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1278..1874 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(1278..1297) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1774..1794 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1884..1940 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 1941..2657 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 1941..2654 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(1983..2004) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2244..2263) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2591..2612 /label=EGFP-C /note="EGFP, forward primer" misc_feature 2673..3261 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2726..2746) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3144..3155) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 3333..3566 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3644..3765 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3681..3700) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3735..3754 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 3805..3940 /label=SV40 ori /note="SV40 origin of replication" primer_bind 3867..3886 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(3960..3979) /label=T7 /note="T7 promoter, forward primer" promoter complement(3961..3979) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3989..4006) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3989..4005) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3998..4020) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4147..4602 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4234..4253) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4444..4465 /label=F1ori-F /note="F1 origin, forward primer" promoter 4628..4732 /gene="bla" /label=AmpR promoter CDS 4733..5593 /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(4951..4970) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5764..6352 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6253..6272 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6506..6523 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6640..6661 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6676..6706 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6714..6730 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 6719..6741 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 6738..6754 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6738..6754 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 6773..6793 /label=T3 /note="T3 promoter, forward primer" promoter 6775..6793 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6821..7047 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 7048..7228 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7275..7400 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7893..8126 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8311..8355 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" promoter 8533..8773 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 8533..8553 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 8704..8723 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 10663..10738 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 10794..10911 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct 61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat 121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt gcctagagaa 181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 361 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc 421 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt 481 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 541 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 601 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 661 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 721 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 781 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 841 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 901 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 961 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 1021 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag 1081 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 1141 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat 1201 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt 1261 cgtgacgtac ggccaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg 1321 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc 1381 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca 1441 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg 1501 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca 1561 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc 1621 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc 1681 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg 1741 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg 1801 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc 1861 gcaagcccgg tgccggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg 1921 tcgaagagaa tcctggaccg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 1981 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 2041 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 2101 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 2161 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 2221 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 2281 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 2341 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 2401 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 2461 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 2521 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 2581 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 2641 acgagctgta caagtaaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg 2701 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 2761 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2821 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2881 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2941 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 3001 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 3061 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 3121 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 3181 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 3241 ccctttgggc cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga 3301 tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag 3361 acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga 3421 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct 3481 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt 3541 ttagtcagtg tggaaaatct ctagcagtac gtatagtagt tcatgtcatc ttattattca 3601 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc 3661 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3721 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctcta 3781 gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 3841 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 3901 gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta cccaattcgc 3961 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg 4021 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 4081 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 4141 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 4201 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 4261 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 4321 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 4381 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 4441 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 4501 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 4561 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg 4621 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 4681 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 4741 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 4801 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 4861 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 4921 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 4981 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 5041 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 5101 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 5161 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 5221 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 5281 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 5341 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 5401 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 5461 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 5521 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 5581 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 5641 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 5701 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 5761 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 5821 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 5881 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 5941 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 6001 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 6061 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 6121 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 6181 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 6241 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 6301 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6361 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 6421 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 6481 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 6541 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 6601 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 6661 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 6721 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa 6781 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc 6841 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag 6901 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa 6961 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta 7021 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg 7081 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 7141 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 7201 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag 7261 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag 7321 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga 7381 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa 7441 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca 7501 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac 7561 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat 7621 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg 7681 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg 7741 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat 7801 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga 7861 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga 7921 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta 7981 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat 8041 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa 8101 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc 8161 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat 8221 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc 8281 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat 8341 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta 8401 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata 8461 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg 8521 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag 8581 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat 8641 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa 8701 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc 8761 ttgtggaaag gacgaaacac cggagacggt tgtaaatgag cacacaaaat acacatgcta 8821 aaatattata ttctatgacc tttataaaat caaccaaaat cttcttttta ataactttag 8881 tatcaataat tagaattttt atgttccttt ttgcaaactt ttaataaaaa tgagcaaaat 8941 aaaaaaacgc tagttttagt aactcgcgtt gttttcttca cctttaataa tagctactcc 9001 accacttgtt cctaagcggt cagctcctgc ttcaatcatt ttttgagcat cttcaaatgt 9061 tctaactcca ccagctgctt taactaaagc attgtcttta acaactgact tcattagttt 9121 aacatcttca aatgttgcac ctgattttga aaatcctgtt gatgttttaa caaattctaa 9181 tccagcttca acagctattt cacaagcttt catgatttct tcttttgtta ataaacaatt 9241 ttccataata catttaacaa catgtgatcc agctgctttt tttacagctt tcatgtcttc 9301 taaaactaat tcataatttt tgtcttttaa tgcaccaata tttaatacca tatcaatttc 9361 tgttgcacca tctttaattg cttcagaaac ttcgaatgct tttgtagctg ttgtgcatgc 9421 acctagagga aaacctacaa catttgttat tcctacattt gtgcctttta ataattcttt 9481 acaatagctt gttcaatatg aattaacaca aactgttgca aaatcaaatt caattgcttc 9541 atcacataat tgtttaattt cagctttcgt agcatcttgt tttaataatg tgtgatctat 9601 atatttgttt agtttcattt tttctcctat atattcattt ttaattttaa ttctttaata 9661 atttcgtcta ctttaacttt agcgttttga acagattcac caacacctat aaaataaatt 9721 tttagtttag gttcagttcc acttgggcga acagcaaatc atgacttatc ttctaaataa 9781 aattttagta agtcttgtcc tggcatatta tacattccat cgatgtagtc ttcaacatta 9841 acaactttaa gtccagcaat ttgagttaag ggtgttgctc tcaatgattt cattaatggt 9901 tcaattttta atttcttttc ttctggttta aaattcaagt ttaaagtgaa agtgtaatat 9961 gcacccattt ctttaaataa atcttctaaa tagtctacta atgttttatt ttgtttttta 10021 taaaatcaag cagcctctgc tattaatata gaagcttgta ttccatcttt atctctagct 10081 gagtcatcaa ttacatatcc ataactttct tcataagcaa aaacaaaatt taatccgtta 10141 tcttcttctt tagcaatttc tctacccatt catttaaatc cagttaaagt ttttacaata 10201 ttaactccat atttttcatg agcgattcta tcacccaaat cacttgttac aaaacttgaa 10261 tatagagccg gattttttgg aatgctattt aagcgtttta gatttgataa ttttcaatca 10321 attaaaattg gtcctgtttg atttccatct aatcttacaa aatgaccatc atgttttatt 10381 gccattccaa atctgtcagc atctgggtca ttcataataa taatatctgc atcatgttta 10441 ataccatatt caagcggtat ttttcatgca ggatcaaatt ctggatttgg atttacaaca 10501 tttttaaatg tttcatcttc aaatgcatgc tcttcaacct caataacgtt atatcctgat 10561 tcacgtaata tttttggggt aaatttagtt cctgttccat taactgcgct aaaaataatt 10621 tttaaatctt ttttagcttc ttgctctttt ttgtacgtct ctgttttaga gctagaaata 10681 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 10741 ttttaagctt ggcgtaacta gatcttgaga caaatggcag tattcatcca caattttaaa 10801 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca 10861 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat 10921 tacagggaca gcagagatcc actttggcgc cggctcgagg ggg //