LOCUS Exported 11571 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION 3rd gen lentiviral expression of ChR2 with mutation fused to EYFP driven by EF1a promoter for optogenetic activation. ACCESSION . VERSION . KEYWORDS plenti2-EF1a-ChR2-EYFP-WRPE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11571) AUTHORS Osaki T, Uzel SGM, Kamm RD TITLE Microphysiological 3D model of amyotrophic lateral sclerosis (ALS) from human iPS-derived muscle cells and optogenetic motor neurons. JOURNAL Sci Adv. 2018 Oct 10;4(10):eaat5847. doi: 10.1126/sciadv.aat5847. eCollection 2018 Oct. PUBMED 30324134 REFERENCE 2 (bases 1 to 11571) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11571 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..66) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 619..817 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 772..792 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /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" misc_feature 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2682..3859 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 2913..3850 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 3807..3827 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" regulatory 3866..3875 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3872..4798 /codon_start=1 /product="H134R mutant of an N-terminal fragment of channelrhodopsin-2 from the alga Chlamydomonas reinhardtii (Nagel et al., 2005)" /label=ChR2(H134R) /note="blue light-activated cation channel" /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI RKTTKLNIGGTEIEVETLVEDEAEAGAV" CDS 4811..6238 /codon_start=1 /product="tandem dimeric (pseudo-monomeric) derivative of DsRed (Shaner et al., 2004)" /label=tdTomato /note="mammalian codon-optimized" /translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH LFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMN FEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVL KGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYER SEGRHHLFLYGMDELYK" misc_feature 6263..6851 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6316..6336) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6734..6745) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(6854..6870) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6855..6871) /label=pBluescriptKS /note="For pBluescript vector" LTR 7376..7556 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(7582..7599) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7588..7812 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 7858..8286 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7945..7964) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8155..8176 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8295..8315) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 8300..8629 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 8480..8615 /label=SV40 ori /note="SV40 origin of replication" primer_bind 8542..8561 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 8677..8724 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 8743..9117 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 9247..9368 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9284..9303) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9338..9357 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(9417..9433) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9417..9433) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(9430..9452) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 9441..9457 /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)." promoter complement(9465..9495) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9510..9531 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(9648..9665) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(9819..10407) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(9899..9918) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(10578..11438) /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 11201..11220 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(11439..11543) /gene="bla" /label=AmpR promoter ORIGIN 1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac 241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat 301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg 361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt 661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct 841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg 1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt 2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat 2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa 2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag 2581 agatccagtt tggttaatta atgcaaagat ggataaagtt ttaaacagag aggaatcttt 2641 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt 2701 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa 2761 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 2821 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 2881 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc 2941 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccact ggctgcagta 3001 cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc 3061 ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg 3121 cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt ctctagccat 3181 ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc 3241 gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg acggggcccg 3301 tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg 3361 acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat 3421 cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag cggaaagatg 3481 gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct cgggagagcg 3541 ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg 3601 tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga gcttttggag 3661 tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc acactgagtg 3721 ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg aatttgccct 3781 ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa gtttttttct 3841 tccatttcag gtgtcgtgac gtacggccac catggactat ggcggcgctt tgtctgccgt 3901 cggacgcgaa cttttgttcg ttactaatcc tgtggtggtg aacgggtccg tcctggtccc 3961 tgaggatcaa tgttactgtg ccggatggat tgaatctcgc ggcacgaacg gcgctcagac 4021 cgcgtcaaat gtcctgcagt ggcttgcagc aggattcagc attttgctgc tgatgttcta 4081 tgcctaccaa acctggaaat ctacatgcgg ctgggaggag atctatgtgt gcgccattga 4141 aatggttaag gtgattctcg agttcttttt tgagtttaag aatccctcta tgctctacct 4201 tgccacagga caccgggtgc agtggctgcg ctatgcagag tggctgctca cttgtcctgt 4261 catccttatc cgcctgagca acctcaccgg cctgagcaac gactacagca ggagaaccat 4321 gggactcctt gtctcagaca tcgggactat cgtgtggggg gctaccagcg ccatggcaac 4381 cggctatgtt aaagtcatct tcttttgtct tggattgtgc tatggcgcga acacattttt 4441 tcacgccgcc aaagcatata tcgagggtta tcatactgtg ccaaagggtc ggtgccgcca 4501 ggtcgtgacc ggcatggcat ggctgttttt cgtgagctgg ggtatgttcc caattctctt 4561 cattttgggg cccgaaggtt ttggcgtcct gagcgtctat ggctccaccg taggtcacac 4621 gattattgat ctgatgagta aaaattgttg ggggttgttg ggacactacc tgcgcgtcct 4681 gatccacgag cacatattga ttcacggaga tatccgcaaa accaccaaac tgaacatcgg 4741 cggaacggag atcgaggtcg agactctcgt cgaagacgaa gccgaggccg gagccgtgcc 4801 agccgcggcc gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg 4861 catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc 4921 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc 4981 ctgggacatc ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc 5041 cgacatcccc gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat 5101 gaacttcgag gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac 5161 gctgatctac aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca 5221 gaagaagacc atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct 5281 gaagggcgag atccaccagg ccctgaagct gaaggacggc ggccactacc tggtggagtt 5341 caagaccatc tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac 5401 caagctggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc 5461 cgagggccgc caccacctgt tcctggggca tggcaccggc agcaccggca gcggcagctc 5521 cggcaccgcc tcctccgagg acaacaacat ggccgtcatc aaagagttca tgcgcttcaa 5581 ggtgcgcatg gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg 5641 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc 5701 cttcgcctgg gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca 5761 ccccgccgac atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg 5821 cgtgatgaac ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga 5881 cggcacgctg atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt 5941 aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg 6001 cgtgctgaag ggcgagatcc accaggccct gaagctgaag gacggcggcc actacctggt 6061 ggagttcaag accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt 6121 ggacaccaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga 6181 gcgctccgag ggccgccacc acctgttcct gtacggcatg gacgagctgt acaagtaaga 6241 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac 6301 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt 6361 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt 6421 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt 6481 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg 6541 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg 6601 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc 6661 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt 6721 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc 6781 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc 6841 cgcctccccg catcgatacc gtcgacctcg agacctagaa aaacatggag caatcacaag 6901 tagcaataca gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggagga 6961 ggtgggtttt ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgt 7021 agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg 7081 aagacaagat atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggca 7141 gaactacaca ccagggccag ggatcagata tccactgacc tttggatggt gctacaagct 7201 agtaccagtt gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgtt 7261 acaccctgtg agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtt 7321 tgacagccgc ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtc 7381 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 7441 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 7501 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggc 7561 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 7621 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 7681 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 7741 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 7801 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg 7861 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 7921 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 7981 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 8041 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat 8101 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 8161 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag 8221 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 8281 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc 8341 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc 8401 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt 8461 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc 8521 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct 8581 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg 8641 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag 8701 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt 8761 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg 8821 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg 8881 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg 8941 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc 9001 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc 9061 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac 9121 gtgctacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 9181 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 9241 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 9301 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 9361 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 9421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 9481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 9541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 9601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 9661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 9721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 9781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 9841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 9901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 9961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 10021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 10081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 10141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 10201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 10261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 10321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 10381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 10441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 10501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 10561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 10621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 10681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 10741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 10801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 10861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 10921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 10981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 11041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 11101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 11161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 11221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 11281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 11341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 11401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 11461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 11521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga c //