LOCUS Exported 13262 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses human codon-optimized inactive SpCas9-NG fused to a transcriptional repressor KRAB in mammalian cells. For cloning of sgRNAs using BsmBI. Contains a barcode downstream of sgRNA cassette.. ACCESSION . VERSION . KEYWORDS pCC_10 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-KRAB-dCas9NG-NLS-2A-... SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 13262) AUTHORS Legut M, Daniloski Z, Xue X, McKenzie D, Guo X, Wessels HH, Sanjana NE TITLE High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation. JOURNAL Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi: 10.1016/j.celrep.2020.02.010. PUBMED 32130891 REFERENCE 2 (bases 1 to 13262) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..13262 /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 2608..2848 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2608..2628 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2779..2798 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 3027..3238 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 3257..3266 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3293..3478 /codon_start=1 /product="Kruppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994)" /label=KRAB /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP" CDS 3500..5218 /codon_start=1 /product="N-terminal portion of Streptococcus pyogenes Cas9 (Zetsche et al., 2015)" /label=Cas9(N) /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE" CDS 7604..7651 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7652..7675 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 7685..7741 /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 7742..8338 /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 8235..8255 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 8354..8942 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8407..8427) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(8825..8836) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 9014..9247 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(9273..9290) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 9279..9503 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 9549..9977 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(9636..9655) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 9846..9867 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(9986..10006) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 9991..10320 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 10171..10306 /label=SV40 ori /note="SV40 origin of replication" primer_bind 10233..10252 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 10368..10415 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 10434..10808 /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 10938..11059 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(10975..10994) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 11029..11048 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(11108..11124) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(11108..11124) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(11121..11143) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 11132..11148 /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(11156..11186) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 11201..11222 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(11339..11356) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(11510..12098) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(11590..11609) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(12269..13129) /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 12892..12911 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(13130..13234) /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 aggtaccgag ggcctatttc ccatgattcc ttcatatttg 2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag 2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc 2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggacgtct ctgtttaaga 2881 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa 2941 gtggcaccga gtcggtgctt tttttgaatt caaattggct agctaggtct tgaaaggagt 3001 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa 3061 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg 3121 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat 3181 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga 3241 ccggttctag agcgctgcca ccatggatgc taagtcacta actgcctggt cccggacact 3301 ggtgaccttc aaggatgtat ttgtggactt caccagggag gagtggaagc tgctggacac 3361 tgctcagcag atcgtgtaca gaaatgtgat gctggagaac tataagaacc tggtttcctt 3421 gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag aagagcccgg 3481 cggttccggc ggagggtcga tggacaagaa gtacagcatc ggcctggcca tcggcaccaa 3541 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt 3601 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggcgccc tgctgttcga 3661 cagcggagaa acagccgagg ccacccggct gaagagaacc gccagaagaa gatacaccag 3721 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga 3781 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga 3841 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac 3901 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat 3961 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct 4021 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca 4081 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc 4141 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa 4201 gaatggcctg ttcggcaacc tgattgccct gagcctgggc ctgaccccca acttcaagag 4261 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga 4321 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa 4381 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa 4441 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct 4501 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca 4561 gagcaagaac ggctacgccg gctacatcga tggcggagcc agccaggaag agttctacaa 4621 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa 4681 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat 4741 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa 4801 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc 4861 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac 4921 cccctggaac ttcgaggaag tggtggacaa gggcgccagc gcccagagct tcatcgagcg 4981 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct 5041 gtacgagtac ttcaccgtgt acaacgagct gaccaaagtg aaatacgtga ccgagggaat 5101 gagaaagccc gccttcctga gcggcgagca gaaaaaagcc atcgtggacc tgctgttcaa 5161 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg 5221 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata 5281 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga 5341 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga 5401 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg 5461 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gggacaagca 5521 gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca gaaacttcat 5581 gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag cccaggtgtc 5641 cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc ccgccattaa 5701 gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga tgggccggca 5761 caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc agaagggaca 5821 gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc tgggcagcca 5881 gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc tgtacctgta 5941 ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca accggctgtc 6001 cgactacgat gtggacgcta tcgtgcctca gagctttctt aaggacgact ccatcgacaa 6061 caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc cctccgaaga 6121 ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc tgattaccca 6181 gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac tggataaggc 6241 cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg tggcacagat 6301 cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc gggaagtgaa 6361 agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc agttttacaa 6421 agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg ccgtcgtggg 6481 aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg gcgactacaa 6541 ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc 6601 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa 6661 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg 6721 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat 6781 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc ggcccaagag 6841 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt 6901 cgttagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc 6961 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt 7021 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct 7081 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct 7141 ggcctctgcc cggttcctgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa 7201 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca 7261 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag 7321 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta 7381 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac 7441 cctgaccaat ctgggagccc ctcgggcctt caagtacttt gacaccacca tcgaccggaa 7501 ggtgtaccgg agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg 7561 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaagcgac ctgccgccac 7621 aaagaaggct ggacaggcta agaagaagaa agattacaaa gacgatgacg ataagggatc 7681 cggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga atcctggacc 7741 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg 7801 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg 7861 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat 7921 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag 7981 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc 8041 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc 8101 cgcgtggttc ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag 8161 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga 8221 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga 8281 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac 8341 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 8401 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 8461 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 8521 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 8581 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 8641 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 8701 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt 8761 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 8821 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 8881 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc 8941 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa 9001 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct 9061 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg 9121 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt 9181 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc 9241 tctagcaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc 9301 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 9361 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 9421 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 9481 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg 9541 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 9601 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 9661 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 9721 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 9781 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 9841 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 9901 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 9961 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca 10021 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt 10081 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca 10141 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc 10201 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct 10261 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa 10321 aagctcccgg gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat 10381 catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca 10441 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct 10501 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc 10561 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc 10621 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt 10681 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg 10741 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc 10801 aggactgaca cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 10861 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 10921 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata 10981 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 11041 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt 11101 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 11161 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 11221 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 11281 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 11341 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 11401 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 11461 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 11521 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 11581 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 11641 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 11701 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 11761 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 11821 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 11881 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 11941 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 12001 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 12061 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 12121 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 12181 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 12241 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 12301 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 12361 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 12421 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 12481 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 12541 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 12601 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 12661 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 12721 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 12781 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 12841 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 12901 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 12961 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 13021 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 13081 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 13141 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 13201 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 13261 ac //