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" LTR 1..181 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 228..353 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 846..1079 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1264..1308 /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 1486..1726 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1486..1506 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1657..1676 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 3616..3691 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 3747..3864 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 4003..5181 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 4234..5172 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 5129..5149 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 5194..5790 /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(5194..5213) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5690..5710 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 5800..5856 /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 5857..6573 /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 5857..6570 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(5899..5920) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6160..6179) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6507..6528 /label=EGFP-C /note="EGFP, forward primer" misc_feature 6589..7177 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6642..6662) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(7060..7071) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 7249..7482 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 7560..7681 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7597..7616) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7651..7670 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 7721..7856 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7783..7802 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(7876..7895) /label=T7 /note="T7 promoter, forward primer" promoter complement(7877..7895) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(7905..7922) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7905..7921) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7914..7936) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 8063..8518 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8150..8169) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8360..8381 /label=F1ori-F /note="F1 origin, forward primer" promoter 8544..8648 /gene="bla" /label=AmpR promoter CDS 8649..9509 /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(8867..8886) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9680..10268 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10169..10188 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 10422..10439 /label=L4440 /note="L4440 vector, forward primer" protein_bind 10556..10577 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 10592..10622 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 10630..10646 /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 10635..10657 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 10654..10670 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 10654..10670 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 10689..10709 /label=T3 /note="T3 promoter, forward primer" promoter 10691..10709 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 10737..10963 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" ORIGIN 1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc 1501 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat 1561 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 1621 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 1681 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac 1741 ggttgtaaat gagcacacaa aatacacatg ctaaaatatt atattctatg acctttataa 1801 aatcaaccaa aatcttcttt ttaataactt tagtatcaat aattagaatt tttatgttcc 1861 tttttgcaaa cttttaataa aaatgagcaa aataaaaaaa cgctagtttt agtaactcgc 1921 gttgttttct tcacctttaa taatagctac tccaccactt gttcctaagc ggtcagctcc 1981 tgcttcaatc attttttgag catcttcaaa tgttctaact ccaccagctg ctttaactaa 2041 agcattgtct ttaacaactg acttcattag tttaacatct tcaaatgttg cacctgattt 2101 tgaaaatcct gttgatgttt taacaaattc taatccagct tcaacagcta tttcacaagc 2161 tttcatgatt tcttcttttg ttaataaaca attttccata atacatttaa caacatgtga 2221 tccagctgct ttttttacag ctttcatgtc ttctaaaact aattcataat ttttgtcttt 2281 taatgcacca atatttaata ccatatcaat ttctgttgca ccatctttaa ttgcttcaga 2341 aacttcgaat gcttttgtag ctgttgtgca tgcacctaga ggaaaaccta caacatttgt 2401 tattcctaca tttgtgcctt ttaataattc tttacaatag cttgttcaat atgaattaac 2461 acaaactgtt gcaaaatcaa attcaattgc ttcatcacat aattgtttaa tttcagcttt 2521 cgtagcatct tgttttaata atgtgtgatc tatatatttg tttagtttca ttttttctcc 2581 tatatattca tttttaattt taattcttta ataatttcgt ctactttaac tttagcgttt 2641 tgaacagatt caccaacacc tataaaataa atttttagtt taggttcagt tccacttggg 2701 cgaacagcaa atcatgactt atcttctaaa taaaatttta gtaagtcttg tcctggcata 2761 ttatacattc catcgatgta gtcttcaaca ttaacaactt taagtccagc aatttgagtt 2821 aagggtgttg ctctcaatga tttcattaat ggttcaattt ttaatttctt ttcttctggt 2881 ttaaaattca agtttaaagt gaaagtgtaa tatgcaccca tttctttaaa taaatcttct 2941 aaatagtcta ctaatgtttt attttgtttt ttataaaatc aagcagcctc tgctattaat 3001 atagaagctt gtattccatc tttatctcta gctgagtcat caattacata tccataactt 3061 tcttcataag caaaaacaaa atttaatccg ttatcttctt ctttagcaat ttctctaccc 3121 attcatttaa atccagttaa agtttttaca atattaactc catatttttc atgagcgatt 3181 ctatcaccca aatcacttgt tacaaaactt gaatatagag ccggattttt tggaatgcta 3241 tttaagcgtt ttagatttga taattttcaa tcaattaaaa ttggtcctgt ttgatttcca 3301 tctaatctta caaaatgacc atcatgtttt attgccattc caaatctgtc agcatctggg 3361 tcattcataa taataatatc tgcatcatgt ttaataccat attcaagcgg tatttttcat 3421 gcaggatcaa attctggatt tggatttaca acatttttaa atgtttcatc ttcaaatgca 3481 tgctcttcaa cctcaataac gttatatcct gattcacgta atatttttgg ggtaaattta 3541 gttcctgttc cattaactgc gctaaaaata atttttaaat cttttttagc ttcttgctct 3601 tttttgtacg tctctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 3661 tcaacttgaa aaagtggcac cgagtcggtg cttttttaag cttggcgtaa ctagatcttg 3721 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt 3781 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa 3841 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg 3901 cgccggctcg agggggcccg ggtgcaaaga tggataaagt tttaaacaga gaggaatctt 3961 tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag 4021 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga 4081 tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc 4141 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt 4201 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg 4261 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag 4321 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc 4381 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc 4441 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc 4501 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat 4561 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc 4621 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc 4681 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt 4741 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga 4801 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag 4861 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca 4921 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg 4981 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag 5041 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc 5101 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt 5161 cttccatttc aggtgtcgtg acgtacggcc accatgaccg agtacaagcc cacggtgcgc 5221 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 5281 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 5341 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 5401 ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 5461 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 5521 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga 5581 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 5641 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 5701 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 5761 acctggtgca tgacccgcaa gcccggtgcc ggatccggcg caacaaactt ctctctgctg 5821 aaacaagccg gagatgtcga agagaatcct ggaccggtga gcaagggcga ggagctgttc 5881 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 5941 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 6001 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 6061 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 6121 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 6181 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 6241 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 6301 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 6361 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 6421 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 6481 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 6541 atcactctcg gcatggacga gctgtacaag taaacgcgtt aagtcgacaa tcaacctctg 6601 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 6661 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 6721 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 6781 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 6841 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 6901 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 6961 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc 7021 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 7081 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 7141 cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt 7201 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 7261 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc 7321 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 7381 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 7441 gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat 7501 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 7561 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 7621 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 7681 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc 7741 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga 7801 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg 7861 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 7921 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 7981 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 8041 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 8101 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 8161 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 8221 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 8281 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 8341 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 8401 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 8461 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 8521 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 8581 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 8641 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 8701 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 8761 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 8821 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 8881 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 8941 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 9001 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 9061 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 9121 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 9181 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 9241 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 9301 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 9361 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 9421 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 9481 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 9541 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 9601 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 9661 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 9721 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 9781 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 9841 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 9901 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 9961 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 10021 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 10081 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 10141 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 10201 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 10261 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 10321 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 10381 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 10441 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 10501 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 10561 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 10621 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 10681 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta 10741 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt 10801 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc 10861 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca 10921 ttgcagagat attgtattta agtgcctagc tcgatacata aac //