LOCUS Exported 11917 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses customizable S. Pyogenes sgRNA from U6 promoter and PuroR-P2A-EGFP from EF-1a promoter. Stuffer contains mRFP from a bacterial promoter, enabling simple visual quality control step of sgRNA. ACCESSION . VERSION . KEYWORDS LentiGuide-Puro-P2A-EGFP_mRFPstuf SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11917) 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 11917) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11917 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 683..713 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 721..737 /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)." RBS 755..766 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 773..1450 /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS TGA" terminator 1495..1566 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" misc_RNA 2844..2919 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 2975..3092 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 3231..4409 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 3462..4400 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 4357..4377 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 4422..5018 /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(4422..4441) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4918..4938 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 5028..5084 /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 5085..5801 /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 5085..5798 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(5127..5148) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5388..5407) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5735..5756 /label=EGFP-C /note="EGFP, forward primer" misc_feature 5817..6405 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5870..5890) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6288..6299) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 6477..6710 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 6788..6909 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6825..6844) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6879..6898 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 6949..7084 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7011..7030 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(7104..7123) /label=T7 /note="T7 promoter, forward primer" promoter complement(7105..7123) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(7133..7150) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(7133..7149) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7142..7164) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 7291..7746 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7378..7397) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7588..7609 /label=F1ori-F /note="F1 origin, forward primer" promoter 7772..7876 /gene="bla" /label=AmpR promoter CDS 7877..8737 /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(8095..8114) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8908..9496 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9397..9416 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9650..9667 /label=L4440 /note="L4440 vector, forward primer" protein_bind 9784..9805 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 9820..9850 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9858..9874 /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 9863..9885 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 9882..9898 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 9882..9898 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 9917..9937 /label=T3 /note="T3 promoter, forward primer" promoter 9919..9937 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 9965..10191 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 10192..10372 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 10419..10544 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 11037..11270 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 11455..11499 /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 11677..11917 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 11677..11697 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 11848..11867 /label=LKO.1 5' /note="Human U6 promoter, forward primer" ORIGIN 1 gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa tattatattc 61 tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat caataattag 121 aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa aaaacgctag 181 ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc acttgttcct 241 aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct aactccacca 301 gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac atcttcaaat 361 gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc agcttcaaca 421 gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc cataatacat 481 ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa aactaattca 541 taatttttgt cttttaatgc accaatattt aataccatat caatttctgt tgcaccatct 601 ttaattgctt cagaaactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 661 tcacacatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 721 ttgtgagcgg ataacaattt cacacatact agagaaagag gagaaatact agatggcttc 781 ctccgaggac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag gttccgttaa 841 cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag gtacccagac 901 cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca tcctgtcccc 961 gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc cggactacct 1021 gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg aggacggtgg 1081 tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct acaaagttaa 1141 actgcgtggt accaacttcc cgtccgatgg tccggttatg cagaaaaaaa ccatgggttg 1201 ggaagcttcc accgaacgta tgtacccgga ggacggtgct ctgaaaggtg aaatcaaaat 1261 gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca cctacatggc 1321 taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg acatcacctc 1381 ccacaacgag gactacacca tcgttgaaca gtacgaacgt gctgaaggtc gtcactccac 1441 cggtgcttaa taacgctgat agtgctagtg tagatcgcta ctagagccag gcatcaaata 1501 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac 1561 gctctctact agagtcacac tggtgtagct gttgtgcatg cacctagagg aaaacctaca 1621 acatttgtta ttcctacatt tgtgcctttt aataattctt tacaatagct tgttcaatat 1681 gaattaacac aaactgttgc aaaatcaaat tcaattgctt catcacataa ttgtttaatt 1741 tcagctttcg tagcatcttg ttttaataat gtgtgatcta tatatttgtt tagtttcatt 1801 ttttctccta tatattcatt tttaatttta attctttaat aatttcgtct actttaactt 1861 tagcgttttg aacagattca ccaacaccta taaaataaat ttttagttta ggttcagttc 1921 cacttgggcg aacagcaaat catgacttat cttctaaata aaattttagt aagtcttgtc 1981 ctggcatatt atacattcca tcgatgtagt cttcaacatt aacaacttta agtccagcaa 2041 tttgagttaa gggtgttgct ctcaatgatt tcattaatgg ttcaattttt aatttctttt 2101 cttctggttt aaaattcaag tttaaagtga aagtgtaata tgcacccatt tctttaaata 2161 aatcttctaa atagtctact aatgttttat tttgtttttt ataaaatcaa gcagcctctg 2221 ctattaatat agaagcttgt attccatctt tatctctagc tgagtcatca attacatatc 2281 cataactttc ttcataagca aaaacaaaat ttaatccgtt atcttcttct ttagcaattt 2341 ctctacccat tcatttaaat ccagttaaag tttttacaat attaactcca tatttttcat 2401 gagcgattct atcacccaaa tcacttgtta caaaacttga atatagagcc ggattttttg 2461 gaatgctatt taagcgtttt agatttgata attttcaatc aattaaaatt ggtcctgttt 2521 gatttccatc taatcttaca aaatgaccat catgttttat tgccattcca aatctgtcag 2581 catctgggtc attcataata ataatatctg catcatgttt aataccatat tcaagcggta 2641 tttttcatgc aggatcaaat tctggatttg gatttacaac atttttaaat gtttcatctt 2701 caaatgcatg ctcttcaacc tcaataacgt tatatcctga ttcacgtaat atttttgggg 2761 taaatttagt tcctgttcca ttaactgcgc taaaaataat ttttaaatct tttttagctt 2821 cttgctcttt tttgtacgtc tctgttttag agctagaaat agcaagttaa aataaggcta 2881 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttagaat tcgcgtaact 2941 agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg 3001 ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat 3061 tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc 3121 cactttggcg ccggctcgag ggggcccggg tgcaaagatg gataaagttt taaacagaga 3181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg 3241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg 3301 gcaattgatc cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt 3361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg 3421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt 3481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct 3541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga 3601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct 3661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt 3721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc 3781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg 3841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac 3901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc 3961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag 4021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct 4081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg 4141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga 4201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc 4261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg 4321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa 4381 gtttttttct tccatttcag gtgtcgtgac gtacggccac catgaccgag tacaagccca 4441 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt 4501 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca 4561 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg 4621 cggacgacgg cgccgccgtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg 4681 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc 4741 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca 4801 ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg 4861 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca 4921 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag 4981 gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg atccggcgca acaaacttct 5041 ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc aagggcgagg 5101 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca 5161 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt 5221 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct 5281 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt 5341 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact 5401 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga 5461 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca 5521 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca 5581 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca 5641 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg 5701 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg 5761 ccgccgggat cactctcggc atggacgagc tgtacaagta aacgcgttaa gtcgacaatc 5821 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt 5881 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg 5941 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 6001 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt 6061 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg 6121 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg 6181 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct 6241 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 6301 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc 6361 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc 6421 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga 6481 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg 6541 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 6601 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 6661 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtacgtatag 6721 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag 6781 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 6841 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 6901 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccat cccgccccta 6961 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca 7021 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga 7081 ggcctaggga cgtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 7141 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 7201 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 7261 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg 7321 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 7381 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 7441 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 7501 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 7561 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 7621 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 7681 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 7741 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 7801 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 7861 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 7921 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 7981 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 8041 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 8101 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 8161 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 8221 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 8281 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 8341 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 8401 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 8461 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 8521 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 8581 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 8641 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 8701 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 8761 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 8821 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 8881 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 8941 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 9001 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 9061 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 9121 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 9181 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 9241 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 9301 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 9361 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 9421 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 9481 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 9541 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 9601 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 9661 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 9721 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 9781 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 9841 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 9901 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc 9961 ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 10021 catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 10081 gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 10141 ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc 10201 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 10261 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 10321 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 10381 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 10441 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 10501 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 10561 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 10621 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 10681 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 10741 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 10801 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 10861 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 10921 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 10981 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 11041 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 11101 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 11161 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 11221 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 11281 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 11341 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 11401 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 11461 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 11521 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 11581 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 11641 tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct 11701 tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta 11761 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 11821 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 11881 ttcgatttct tggctttata tatcttgtgg aaaggac //