LOCUS Exported 11734 bp ds-DNA circular SYN 09-DEC-2021 DEFINITION Expresses RfxCas13d in mammalian cells, localized in the nucleus. For cloning of guide RNAs compatible with RfxCas13d. Contains a 5' direct repeat. Clone using BsmBI. F overhang aaac. R overhang aaaa. ACCESSION . VERSION . KEYWORDS pLentiRNACRISPR_005 - hU6-DR_BsmBI-EFS-RfxCas13d-NLS-2A-Puro-WPRE SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11734) AUTHORS Wessels HH, Mendez-Mancilla A, Guo X, Legut M, Daniloski Z, Sanjana NE TITLE Massively parallel Cas13 screens reveal principles for guide RNA design. JOURNAL Nat Biotechnol. 2020 Jun;38(6):722-727. doi: 10.1038/s41587-020-0456-9. Epub 2020 Mar 16. PUBMED 32518401 REFERENCE 2 (bases 1 to 11734) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 9, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11734 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 382..580 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 535..555 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 598..778 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 825..950 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1443..1676 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1861..1905 /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 2203..2320 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2371..2611 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2371..2391 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2542..2561 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 2621..2650 /label=RfxCas13d DR30 /note="5' processed 30-base direct repeat for an RfxCas13d gRNA (Konermann et al., 2018)" promoter 2729..2940 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 2965..5862 /codon_start=1 /product="RNA-targeting Type VI CRISPR protein from Ruminococcus flavefaciens strain XPD3002 (Konermann et al., 2018)" /label=RfxCas13d /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII NYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVH AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGN" CDS 5863..5910 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5920..5976 /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 5977..6573 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 6470..6490 /label=Puro-F /note="Puromycin resistance gene, 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" primer_bind complement(7508..7525) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 7514..7738 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 7784..8212 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7871..7890) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8081..8102 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(8221..8241) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 8226..8555 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 8406..8541 /label=SV40 ori /note="SV40 origin of replication" primer_bind 8468..8487 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 8603..8650 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 8669..9043 /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 9173..9294 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(9210..9229) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 9264..9283 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(9343..9359) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9343..9359) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(9356..9378) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 9367..9383 /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(9391..9421) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9436..9457 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(9574..9591) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(9745..10333) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(9825..9844) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(10504..11364) /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 11127..11146 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(11365..11469) /gene="bla" /label=AmpR promoter primer_bind complement(11544..11563) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg 601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga 841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat 1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2341 cagagatcca gtttggttaa ttaaggtacc gagggcctat ttcccatgat tccttcatat 2401 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca 2461 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt 2521 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat 2581 ttcttggctt tatatatctt gtggaaagga cgaaacaccg aacccctacc aactggtcgg 2641 ggtttgaaac ggagacggac gtctcttttt tttgaattcg ctagctaggt cttgaaagga 2701 gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 2761 aagttggggg gaggggtcgg caattgatcc ggtgcctaga gaaggtggcg cggggtaaac 2821 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 2881 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 2941 gaccggttct agagcgctgc caccatgatc gaaaaaaaaa agtccttcgc caagggcatg 3001 ggcgtgaagt ccacactcgt gtccggctcc aaagtgtaca tgacaacctt cgccgaaggc 3061 agcgacgcca ggctggaaaa gatcgtggag ggcgacagca tcaggagcgt gaatgagggc 3121 gaggccttca gcgctgaaat ggccgataaa aacgccggct ataagatcgg caacgccaaa 3181 ttcagccatc ctaagggcta cgccgtggtg gctaacaacc ctctgtatac aggacccgtc 3241 cagcaggata tgctcggcct gaaggaaact ctggaaaaga ggtacttcgg cgagagcgct 3301 gatggcaatg acaatatttg tatccaggtg atccataaca tcctggacat tgaaaaaatc 3361 ctcgccgaat acattaccaa cgccgcctac gccgtcaaca atatctccgg cctggataag 3421 gacattattg gattcggcaa gttctccaca gtgtatacct acgacgaatt caaagacccc 3481 gagcaccata gggccgcttt caacaataac gataagctca tcaacgccat caaggcccag 3541 tatgacgagt tcgacaactt cctcgataac cccagactcg gctatttcgg ccaggccttt 3601 ttcagcaagg agggcagaaa ttacatcatc aattacggca acgaatgcta tgacattctg 3661 gccctcctga gcggactgag gcactgggtg gtccataaca acgaagaaga gtccaggatc 3721 tccaggacct ggctctacaa cctcgataag aacctcgaca acgaatacat ctccaccctc 3781 aactacctct acgacaggat caccaatgag ctgaccaact ccttctccaa gaactccgcc 3841 gccaacgtga actatattgc cgaaactctg ggaatcaacc ctgccgaatt cgccgaacaa 3901 tatttcagat tcagcattat gaaagagcag aaaaacctcg gattcaatat caccaagctc 3961 agggaagtga tgctggacag gaaggatatg tccgagatca ggaaaaatca taaggtgttc 4021 gactccatca ggaccaaggt ctacaccatg atggactttg tgatttatag gtattacatc 4081 gaagaggatg ccaaggtggc tgccgccaat aagtccctcc ccgataatga gaagtccctg 4141 agcgagaagg atatctttgt gattaacctg aggggctcct tcaacgacga ccagaaggat 4201 gccctctact acgatgaagc taatagaatt tggagaaagc tcgaaaatat catgcacaac 4261 atcaaggaat ttaggggaaa caagacaaga gagtataaga agaaggacgc ccctagactg 4321 cccagaatcc tgcccgctgg ccgtgatgtt tccgccttca gcaaactcat gtatgccctg 4381 accatgttcc tggatggcaa ggagatcaac gacctcctga ccaccctgat taataaattc 4441 gataacatcc agagcttcct gaaggtgatg cctctcatcg gagtcaacgc taagttcgtg 4501 gaggaatacg cctttttcaa agactccgcc aagatcgccg atgagctgag gctgatcaag 4561 tccttcgcta gaatgggaga acctattgcc gatgccagga gggccatgta tatcgacgcc 4621 atccgtattt taggaaccaa cctgtcctat gatgagctca aggccctcgc cgacaccttt 4681 tccctggacg agaacggaaa caagctcaag aaaggcaagc acggcatgag aaatttcatt 4741 attaataacg tgatcagcaa taaaaggttc cactacctga tcagatacgg tgatcctgcc 4801 cacctccatg agatcgccaa aaacgaggcc gtggtgaagt tcgtgctcgg caggatcgct 4861 gacatccaga aaaaacaggg ccagaacggc aagaaccaga tcgacaggta ctacgaaact 4921 tgtatcggaa aggataaggg caagagcgtg agcgaaaagg tggacgctct cacaaagatc 4981 atcaccggaa tgaactacga ccaattcgac aagaaaagga gcgtcattga ggacaccggc 5041 agggaaaacg ccgagaggga gaagtttaaa aagatcatca gcctgtacct caccgtgatc 5101 taccacatcc tcaagaatat tgtcaatatc aacgccaggt acgtcatcgg attccattgc 5161 gtcgagcgtg atgctcaact gtacaaggag aaaggctacg acatcaatct caagaaactg 5221 gaagagaagg gattcagctc cgtcaccaag ctctgcgctg gcattgatga aactgccccc 5281 gataagagaa aggacgtgga aaaggagatg gctgaaagag ccaaggagag cattgacagc 5341 ctcgagagcg ccaaccccaa gctgtatgcc aattacatca aatacagcga cgagaagaaa 5401 gccgaggagt tcaccaggca gattaacagg gagaaggcca aaaccgccct gaacgcctac 5461 ctgaggaaca ccaagtggaa tgtgatcatc agggaggacc tcctgagaat tgacaacaag 5521 acatgtaccc tgttcagaaa caaggccgtc cacctggaag tggccaggta tgtccacgcc 5581 tatatcaacg acattgccga ggtcaattcc tacttccaac tgtaccatta catcatgcag 5641 agaattatca tgaatgagag gtacgagaaa agcagcggaa aggtgtccga gtacttcgac 5701 gctgtgaatg acgagaagaa gtacaacgat aggctcctga aactgctgtg tgtgcctttc 5761 ggctactgta tccccaggtt taagaacctg agcatcgagg ccctgttcga taggaacgag 5821 gccgccaagt tcgacaagga gaaaaagaag gtgtccggca ataagcgacc tgccgccaca 5881 aagaaggctg gacaggctaa gaagaagaaa ggatccggcg caacaaactt ctctctgctg 5941 aaacaagccg gagatgtcga agagaatcct ggaccgaccg agtacaagcc cacggtgcgc 6001 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac 6061 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg 6121 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 6181 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 6241 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 6301 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcgga 6361 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 6421 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 6481 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 6541 acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt 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 cagggcccgt ttaaacccgc 7501 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 7561 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 7621 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 7681 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct 7741 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc 7801 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 7861 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 7921 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 7981 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 8041 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 8101 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 8161 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt 8221 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 8281 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 8341 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 8401 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 8461 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 8521 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca 8581 ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata tcggcatagt 8641 ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct 8701 caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg 8761 ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag 8821 cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct 8881 ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg 8941 gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc 9001 ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc tacgagattt 9061 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg 9121 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt 9181 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc 9241 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt 9301 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt 9361 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 9421 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 9481 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 9541 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 9601 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 9661 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 9721 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 9781 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 9841 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 9901 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 9961 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 10021 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 10081 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 10141 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 10201 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 10261 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 10321 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 10381 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 10441 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 10501 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 10561 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 10621 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 10681 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 10741 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 10801 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 10861 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 10921 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 10981 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 11041 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 11101 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 11161 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 11221 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 11281 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 11341 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 11401 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 11461 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc 11521 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 11581 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca 11641 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 11701 ctgcttcgcg atgtacgggc cagatatacg cgtt //