Dean22April Posted November 27, 2017 Posted November 27, 2017 Hi! So I'm in a group testing whether there has been CRISPR/Cas9 editing in 36 wheat plants and we discovered the following (see picture attached): that some plants had high Cas9 expression and no guide RNA expression, and some plants had high guide RNA expression and no Cas9 expression (Cas9 +guide RNA 2 components of CRISPR needed for editing of genes). I am really perplexed why this may be (trend is noted especially in plants 3 and 4) especially as guide RNAs and the Cas9 gene were transformed in equal quantities, is there any idea? Or any idea how we could re-transform plants so this doesn't happen? Thank you so much for any help!
Vmedvil Posted November 27, 2017 Posted November 27, 2017 8 minutes ago, Dean22April said: Hi! So I'm in a group testing whether there has been CRISPR/Cas9 editing in 36 wheat plants and we discovered the following (see picture attached): that some plants had high Cas9 expression and no guide RNA expression, and some plants had high guide RNA expression and no Cas9 expression (Cas9 +guide RNA 2 components of CRISPR needed for editing of genes). I am really perplexed why this may be (trend is noted especially in plants 3 and 4) especially as guide RNAs and the Cas9 gene were transformed in equal quantities, is there any idea? Or any idea how we could re-transform plants so this doesn't happen? Thank you so much for any help! What method did you use the first time to insert both the RNA and CRISPR
Dean22April Posted November 27, 2017 Author Posted November 27, 2017 45 minutes ago, Vmedvil said: What method did you use the first time to insert both the RNA and CRISPR We used biolistic mediated-transformation using tungsten rings coated in DNA fragments delivered directly to developing wheat embryos
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 10 minutes ago, Dean22April said: We used biolistic mediated-transformation using tungsten rings coated in DNA fragments delivered directly to developing wheat embryos Seriously Try Virus Mediated you will be amazed when that doesn't happen but PEG sucks for exacting results, but virii don't that is somewhere in-between, biolistic or even bacterial plasmid is slightly better. The DNA when forced into the cell didn't get sent to every cell because of different in densities of DNA fragments as they traveled into them. Edited November 27, 2017 by Vmedvil
Dean22April Posted November 27, 2017 Author Posted November 27, 2017 (edited) 14 minutes ago, Vmedvil said: Seriously Try Virus Mediated you will be amazed when that doesn't happen but PEG sucks for exacting results, but virii don't that is somewhere in-between, biolistic. Hey, Vmedvil, thanks for your help, I'll look into that as it sounds really good. We'll probably do this next year as still waiting on NGS results to see if there have been edits at all using the current methods but I'm going to suggest to my supervisor we try this as a next step after all our analysis of the current results 14 minutes ago, Vmedvil said: Seriously Try Virus Mediated you will be amazed when that doesn't happen but PEG sucks for exacting results, but virii don't that is somewhere in-between, biolistic or even bacterial plasmid is slightly better. The DNA when forced into the cell didn't get sent to every cell because of different in densities of DNA fragments as they traveled into them. Do you know if this would work on plants? I've just quickly looked it up on google scholar and all the examples seem to be in mammalian cells? Thanks Would gemini-virus replicons work? Edited November 27, 2017 by Dean22April
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 10 minutes ago, Dean22April said: Hey, Vmedvil, thanks for your help, I'll look into that as it sounds really good. We'll probably do this next year as still waiting on NGS results to see if there have been edits at all using the current methods but I'm going to suggest to my supervisor we try this as a next step after all our analysis of the current results Do you know if this would work on plants? I've just quickly looked it up on google scholar and all the examples seem to be in mammalian cells? Thanks You have to use a different virus type, viruses use glycoproteins to target the cell, you will have to find a virus base for the vector that is lysogenic and also has glycoproteins that target your plant cells type or I can. NBCI Edited November 27, 2017 by Vmedvil
Dean22April Posted November 27, 2017 Author Posted November 27, 2017 2 minutes ago, Vmedvil said: You have to use a different virus type, viruses use glycoproteins to target the cell, you will have to find a virus base that is lysogenic and also has glycoproteins that target your plant cells type or I can. NBCI Oh okay, I may ask for help on this as I know very little about this area of research. I'm working on wheat (triticum aestivium cv cadenza) so not sure what glycoproteins target that cell, I'm researching now
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 27 minutes ago, Dean22April said: Oh okay, I may ask for help on this as I know very little about this area of research. I'm working on wheat (triticum aestivium cv cadenza) so not sure what glycoproteins target that cell, I'm researching now See, I did some pre-reasrch on this wheat yellow mosaic virus is what I was thinking but I dunno if it is lysogenic yet. I know most of the other plant mosaics are but not if this one is, which it is just like HIV for humans. "positive-stranded RNA virus replication model. Positive-stranded RNA virus transcription is the method of transcription" So, it has a Reverse transcriptase protein in it, ya, it is basically a plant Retrovirus virus. Genome and Protonomics Yellow Wheat Mosaic Virus You going to want to put UTR 3' - DNA insert - UTR 5' then remove it from the Natural of the Virus code, that turns it into a Plant Retroviral Vector then it will not transfer the proteins necessary for self replication. So it will look like this. Yellow Wheat Mosaic Vector tatggcatcc 181 accagttcta acacttacta catggatggt gaccctagat ggccgacgcg agacccagga 241 gcccctatta ctagggacgc tctgccacag cgcatatctg aagcttggaa caccgtcatc 301 ataaggcaca tgctttctga tggcgatgac caagactcca ttcttggcag agatggttta 361 ccagcaacac gattcaacgc ttacagtggc ttgctgccaa ccttcgttca atctcttggt 421 ctccctgtta accgcttaag gcttcatgca cctgtatcag caattgaagc cccgctatgc 481 gtagacacct cctatgcgcc atggttatac atgtccaatg gaacccacgc atatgaagca 541 acaagattac aaccagttcg gacctttatt gctttcaact tcgcaaacgg ctactgctat 601 ctgaactttt tcattccaat gagctttcag atcagtggcg ccaacgttga agccttctcc 661 cgattcatcg agcaacttcc tgaggtgctt ggcgcatacc caacgcttgg aaacctcttg 721 aaaacagcca tctacttaat gcgcatcttc ccggaaatct tggacgcccc aatccctatc 781 atcgcgaagc gtccaggtgt tgcgcagttt cacgtaacag ataacagagg tttacctcca 841 acatggtttt ccatgatgtg tggtagtgtc tcatcgttcg tgacgctctt actgcacaat 901 cttggcaatg aactcctcaa cggcattgtc ggctcagctg aggaaacagg tcaatacaca 961 aattggaatt ctgaacatga ccactggatt acaagcaggt ttataactct cgaagattac 1021 tacaccacga tgtcctcagc tttgagcgtt gattatcgaa caaaaggtgg ttgcgcagcc 1081 ctctacgacc tgttttcaga tcttggctat agtaatatgg tccggcgaac aaaacgcttt 1141 ccagagagcg cgcaaggttt gagtagcttc tacttccacc tctccgacag agcaccattt 1201 gaagctatcc aagtttttct cagtgttttg cgcgaaatca tccacagcga tgactacgac 1261 tcaccataca ggagcatccg agctcggctt atcaatctta gtagtcttcc ttatgacaat 1321 ccagatgcat gcctcacacg gaacatcttc gaagacgaga ataagcttgt ttggaacttc 1381 gagttttaca aagtcatgac tatagccaaa aacgccactg ctgatcggga gatcttcttc 1441 aggagtcatg ttctaccttt cagatccttt gttaaagaag cttccaatga tcgatacaac 1501 ctgccaagcc catttgagat aacaacgcct gtgaaagcca ctcccgacga ggttcccctt 1561 gttgaaagcg cgactgaatc tacaaccgca aatctctcag catggtggca ggttgcgatg 1621 ggcttaatca ctaccattct tgcagcactc cttgtcttct tctggcgttg tttccttagt 1681 gcaaaaaaga ttaagttccg aaagaaagac acctttccat ggttcggcta ctcccacgga 1741 ggtcctccac cttcaccacc aggtgggcca cccccaggtt caccacggga tgcttcttac 1801 cagcaagttc cacgcactgt tgttcgtgac ctcagctttg atgaggacga tgatctacag 1861 agcgttggct tggaggaagc aggaattcgc ttcaagagtt tgatcaccac tatcgaaaga 1921 ggaaatctcc aagagcttca agcagtcatt ccagagcaca ttagtgatct caatgttctg 1981 cagtcgagcg cccatggatc tggtttctac actatggtgt cactatactt gtccacccta 2041 ggtgatgcaa taacagcttt tgagcagagg aatgatgtct caccagcaac aattcagtcg 2101 cttagaactc tcgaactcca actcgaagct cgccatctca gatttaatga agctggaact 2161 ccaacacaca tcctccagag gagcatcagc gcctctgttg ggcgagcaat tatcagatta 2221 actcaaagcg ccctccttgc atctggcgag gggtttcgaa cacgaatggc atccacgctg 2281 caacgaattg ctgatgagag atctaacaac cttacctcct tcgacgcaag agcactggac 2341 atgacgagcg agcttttcca atccatagaa gccgcccttg actctgacag tagtgatatt 2401 gtctcgctgc ttgctaatgc tgaggcctcg ctgcaggtgt acaacaactt cttcggggtt 2461 aactacgttt cgacgacgct tcttgcactt agacgcgagc taattctccg aagcgcagaa 2521 gggcgcgtcg gtgaacaacc aactggaatc tccgaagagt caaatgaaga gcttgtgcaa 2581 aaatcaatgc aaaaactaga taaagaaatc gagctcttcc aggcccagat cgacagtcag 2641 cgacgagttg cagagataac cgaatcttca aacctaaggg aaaacatcct gcaaccaata 2701 aacactgtag caaatatagc catggcagga gctttcttaa gaggtggagc acggagtcga 2761 ctcccaggtg ttctccccca agcacaaccc cacacacaag cgtttcgccc attcacgggc 2821 cgggcgcatc acctcaccac aacaggacgg gtccagcgct tcctcaggag gccgggccat 2881 tagttttccg catttgctca ttcgcacctc gcataggccg ccctactttc aacgctttct 2941 ttattacttt ggtcattaat atgatcaggc acacactcat cgtggcgccc cattgtctcc 3001 acaatagttt aaattgagca acccaaaaca ccaaaaatac catcatgcat tttgtacttc 3061 catctgcatt ccaccccatt gtgtttagct aatcgtttcg tcatcaaagt atggcgagtg 3121 taaactccac atctgtcggc ttgaccgagt gtatgggacc ttcgacaaaa ttgttacgag 3181 ttgt Transfer Plasmid 1 aaaataaaac caccacaaac aaaaccgacc aaagctatca caaacccaca cacactgctc 61 tttcatctga gcgcatagct tttctctgtc ttcacccgcg cgtgtccgtc cctcctgagc 121 cgtagccttg attccatctt gtgcttcgtt ctcctagttc tttccgcttc (Your DNA Fragments) atacct ggctctagac ttaaaccttt agagcttggg gcgaacttgg aagatagcac 3241 actacttgcg cctataaagc ccaactgtct gtgacccttc acaacgattt ctagatgatt 3301 ctacggaatt agtctttcaa atatctcggc ggcactcgac acgattgcat ccacaacaca 3361 acgcaaagtc cggtttggat atctcgaact ctagttacgg tacgtcatgg agcaacctgg 3421 cggccctacg tcatgatact tcgagctcta cctctttagc accgcgcgtt gtgccacgtc 3481 attaaacccc cagcatgttt attttctgtc gtcccgatga agctttcgag ccaagggaca 3541 cctgcataga aaacgtcact cttgtgatgc aaaaacaatg cacctgtcga tcgtcattga 3601 tcggcggctg ttgtttgaca aacccagttt ttgtccccag gaaatgtgcc Edited November 27, 2017 by Vmedvil
Dean22April Posted November 27, 2017 Author Posted November 27, 2017 25 minutes ago, Vmedvil said: See, I did some pre-reasrch on this wheat yellow mosaic virus is what I was thinking but I dunno if it is lysogenic yet. I know most of the other plant mosaics are but not if this one is, which it is just like HIV for humans. "positive-stranded RNA virus replication model. Positive-stranded RNA virus transcription is the method of transcription" So, it has a Reverse transcriptase protein in it, ya, it is basically a plant Retrovirus virus. Genome and Protonomics Yellow Wheat Mosaic Virus You going to want to put UTR 3' - DNA insert - UTR 5' then remove it from the Natural of the Virus code, that turns it into a Plant Retroviral Vector then it will not transfer the proteins necessary for self replication. So it will look like this. Yellow Wheat Mosaic Vector tatggcatcc 181 accagttcta acacttacta catggatggt gaccctagat ggccgacgcg agacccagga 241 gcccctatta ctagggacgc tctgccacag cgcatatctg aagcttggaa caccgtcatc 301 ataaggcaca tgctttctga tggcgatgac caagactcca ttcttggcag agatggttta 361 ccagcaacac gattcaacgc ttacagtggc ttgctgccaa ccttcgttca atctcttggt 421 ctccctgtta accgcttaag gcttcatgca cctgtatcag caattgaagc cccgctatgc 481 gtagacacct cctatgcgcc atggttatac atgtccaatg gaacccacgc atatgaagca 541 acaagattac aaccagttcg gacctttatt gctttcaact tcgcaaacgg ctactgctat 601 ctgaactttt tcattccaat gagctttcag atcagtggcg ccaacgttga agccttctcc 661 cgattcatcg agcaacttcc tgaggtgctt ggcgcatacc caacgcttgg aaacctcttg 721 aaaacagcca tctacttaat gcgcatcttc ccggaaatct tggacgcccc aatccctatc 781 atcgcgaagc gtccaggtgt tgcgcagttt cacgtaacag ataacagagg tttacctcca 841 acatggtttt ccatgatgtg tggtagtgtc tcatcgttcg tgacgctctt actgcacaat 901 cttggcaatg aactcctcaa cggcattgtc ggctcagctg aggaaacagg tcaatacaca 961 aattggaatt ctgaacatga ccactggatt acaagcaggt ttataactct cgaagattac 1021 tacaccacga tgtcctcagc tttgagcgtt gattatcgaa caaaaggtgg ttgcgcagcc 1081 ctctacgacc tgttttcaga tcttggctat agtaatatgg tccggcgaac aaaacgcttt 1141 ccagagagcg cgcaaggttt gagtagcttc tacttccacc tctccgacag agcaccattt 1201 gaagctatcc aagtttttct cagtgttttg cgcgaaatca tccacagcga tgactacgac 1261 tcaccataca ggagcatccg agctcggctt atcaatctta gtagtcttcc ttatgacaat 1321 ccagatgcat gcctcacacg gaacatcttc gaagacgaga ataagcttgt ttggaacttc 1381 gagttttaca aagtcatgac tatagccaaa aacgccactg ctgatcggga gatcttcttc 1441 aggagtcatg ttctaccttt cagatccttt gttaaagaag cttccaatga tcgatacaac 1501 ctgccaagcc catttgagat aacaacgcct gtgaaagcca ctcccgacga ggttcccctt 1561 gttgaaagcg cgactgaatc tacaaccgca aatctctcag catggtggca ggttgcgatg 1621 ggcttaatca ctaccattct tgcagcactc cttgtcttct tctggcgttg tttccttagt 1681 gcaaaaaaga ttaagttccg aaagaaagac acctttccat ggttcggcta ctcccacgga 1741 ggtcctccac cttcaccacc aggtgggcca cccccaggtt caccacggga tgcttcttac 1801 cagcaagttc cacgcactgt tgttcgtgac ctcagctttg atgaggacga tgatctacag 1861 agcgttggct tggaggaagc aggaattcgc ttcaagagtt tgatcaccac tatcgaaaga 1921 ggaaatctcc aagagcttca agcagtcatt ccagagcaca ttagtgatct caatgttctg 1981 cagtcgagcg cccatggatc tggtttctac actatggtgt cactatactt gtccacccta 2041 ggtgatgcaa taacagcttt tgagcagagg aatgatgtct caccagcaac aattcagtcg 2101 cttagaactc tcgaactcca actcgaagct cgccatctca gatttaatga agctggaact 2161 ccaacacaca tcctccagag gagcatcagc gcctctgttg ggcgagcaat tatcagatta 2221 actcaaagcg ccctccttgc atctggcgag gggtttcgaa cacgaatggc atccacgctg 2281 caacgaattg ctgatgagag atctaacaac cttacctcct tcgacgcaag agcactggac 2341 atgacgagcg agcttttcca atccatagaa gccgcccttg actctgacag tagtgatatt 2401 gtctcgctgc ttgctaatgc tgaggcctcg ctgcaggtgt acaacaactt cttcggggtt 2461 aactacgttt cgacgacgct tcttgcactt agacgcgagc taattctccg aagcgcagaa 2521 gggcgcgtcg gtgaacaacc aactggaatc tccgaagagt caaatgaaga gcttgtgcaa 2581 aaatcaatgc aaaaactaga taaagaaatc gagctcttcc aggcccagat cgacagtcag 2641 cgacgagttg cagagataac cgaatcttca aacctaaggg aaaacatcct gcaaccaata 2701 aacactgtag caaatatagc catggcagga gctttcttaa gaggtggagc acggagtcga 2761 ctcccaggtg ttctccccca agcacaaccc cacacacaag cgtttcgccc attcacgggc 2821 cgggcgcatc acctcaccac aacaggacgg gtccagcgct tcctcaggag gccgggccat 2881 tagttttccg catttgctca ttcgcacctc gcataggccg ccctactttc aacgctttct 2941 ttattacttt ggtcattaat atgatcaggc acacactcat cgtggcgccc cattgtctcc 3001 acaatagttt aaattgagca acccaaaaca ccaaaaatac catcatgcat tttgtacttc 3061 catctgcatt ccaccccatt gtgtttagct aatcgtttcg tcatcaaagt atggcgagtg 3121 taaactccac atctgtcggc ttgaccgagt gtatgggacc ttcgacaaaa ttgttacgag 3181 ttgt Transfer Plasmid 1 aaaataaaac caccacaaac aaaaccgacc aaagctatca caaacccaca cacactgctc 61 tttcatctga gcgcatagct tttctctgtc ttcacccgcg cgtgtccgtc cctcctgagc 121 cgtagccttg attccatctt gtgcttcgtt ctcctagttc tttccgcttc (Your DNA Fragments) atacct ggctctagac ttaaaccttt agagcttggg gcgaacttgg aagatagcac 3241 actacttgcg cctataaagc ccaactgtct gtgacccttc acaacgattt ctagatgatt 3301 ctacggaatt agtctttcaa atatctcggc ggcactcgac acgattgcat ccacaacaca 3361 acgcaaagtc cggtttggat atctcgaact ctagttacgg tacgtcatgg agcaacctgg 3421 cggccctacg tcatgatact tcgagctcta cctctttagc accgcgcgtt gtgccacgtc 3481 attaaacccc cagcatgttt attttctgtc gtcccgatga agctttcgag ccaagggaca 3541 cctgcataga aaacgtcact cttgtgatgc aaaaacaatg cacctgtcga tcgtcattga 3601 tcggcggctg ttgtttgaca aacccagttt ttgtccccag gaaatgtgcc Oh wow! Honestly thank you so much for all this help that is amazing!! I'll take this to my supervisor meeting and now start some more research on this. Thanks again!!
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 20 minutes ago, Dean22April said: Oh wow! Honestly thank you so much for all this help that is amazing!! I'll take this to my supervisor meeting and now start some more research on this. Thanks again!! I want to warn you don't alter that code if you don't know what your doing, we don't want a genetically modified wheat plague and it will only transfect wheat. Edited November 27, 2017 by Vmedvil
Dean22April Posted November 27, 2017 Author Posted November 27, 2017 26 minutes ago, Vmedvil said: I want to warn you don't alter that code if you don't know what your doing, we don't want a genetically modified wheat plague and it will only transfect wheat. Ahaha, thanks for the warning! I'll try not to create a dangerous new super-mutant aha!
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 58 minutes ago, Dean22April said: Ahaha, thanks for the warning! I'll try not to create a dangerous new super-mutant aha! Ya, not responsible if your wheat plant turns into something like this. I will say Remember when I said not to screw with that Potyviridae Viral Vector's code, which should be stable like that. 58 minutes ago, Dean22April said: Ahaha, thanks for the warning! I'll try not to create a dangerous new super-mutant aha! Which I am absolutely not certain if it needs RNA 1 and RNA 2 to construct itself, It may not function properly with just RNA 2 not used to plant viruses. It looks to small to be complete, the length of its genome at around 3000, like HIV-1 is around 9600 Bp. Edited November 27, 2017 by Vmedvil
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 2 hours ago, Dean22April said: Oh wow! Honestly thank you so much for all this help that is amazing!! I'll take this to my supervisor meeting and now start some more research on this. Thanks again!! Ya, I don't if it works or not the Chinese genetics team did a bad job of labeling which I am noticing is still better than the others, the LTR should be where those UTR's are but I am not certain along with it is split in RNA 1 and 2, so I dunno if it will function like that with just RNA 2. RNA 1 YMWV RNA 2 YMWV See the machinery I use is not split like that. 3 hours ago, Vmedvil said: See, I did some pre-reasrch on this wheat yellow mosaic virus is what I was thinking but I dunno if it is lysogenic yet. I know most of the other plant mosaics are but not if this one is, which it is just like HIV for humans. "positive-stranded RNA virus replication model. Positive-stranded RNA virus transcription is the method of transcription" So, it has a Reverse transcriptase protein in it, ya, it is basically a plant Retrovirus virus. Genome and Protonomics Yellow Wheat Mosaic Virus You going to want to put UTR 3' - DNA insert - UTR 5' then remove it from the Natural of the Virus code, that turns it into a Plant Retroviral Vector then it will not transfer the proteins necessary for self replication. So it will look like this. Yellow Wheat Mosaic Vector tatggcatcc 181 accagttcta acacttacta catggatggt gaccctagat ggccgacgcg agacccagga 241 gcccctatta ctagggacgc tctgccacag cgcatatctg aagcttggaa caccgtcatc 301 ataaggcaca tgctttctga tggcgatgac caagactcca ttcttggcag agatggttta 361 ccagcaacac gattcaacgc ttacagtggc ttgctgccaa ccttcgttca atctcttggt 421 ctccctgtta accgcttaag gcttcatgca cctgtatcag caattgaagc cccgctatgc 481 gtagacacct cctatgcgcc atggttatac atgtccaatg gaacccacgc atatgaagca 541 acaagattac aaccagttcg gacctttatt gctttcaact tcgcaaacgg ctactgctat 601 ctgaactttt tcattccaat gagctttcag atcagtggcg ccaacgttga agccttctcc 661 cgattcatcg agcaacttcc tgaggtgctt ggcgcatacc caacgcttgg aaacctcttg 721 aaaacagcca tctacttaat gcgcatcttc ccggaaatct tggacgcccc aatccctatc 781 atcgcgaagc gtccaggtgt tgcgcagttt cacgtaacag ataacagagg tttacctcca 841 acatggtttt ccatgatgtg tggtagtgtc tcatcgttcg tgacgctctt actgcacaat 901 cttggcaatg aactcctcaa cggcattgtc ggctcagctg aggaaacagg tcaatacaca 961 aattggaatt ctgaacatga ccactggatt acaagcaggt ttataactct cgaagattac 1021 tacaccacga tgtcctcagc tttgagcgtt gattatcgaa caaaaggtgg ttgcgcagcc 1081 ctctacgacc tgttttcaga tcttggctat agtaatatgg tccggcgaac aaaacgcttt 1141 ccagagagcg cgcaaggttt gagtagcttc tacttccacc tctccgacag agcaccattt 1201 gaagctatcc aagtttttct cagtgttttg cgcgaaatca tccacagcga tgactacgac 1261 tcaccataca ggagcatccg agctcggctt atcaatctta gtagtcttcc ttatgacaat 1321 ccagatgcat gcctcacacg gaacatcttc gaagacgaga ataagcttgt ttggaacttc 1381 gagttttaca aagtcatgac tatagccaaa aacgccactg ctgatcggga gatcttcttc 1441 aggagtcatg ttctaccttt cagatccttt gttaaagaag cttccaatga tcgatacaac 1501 ctgccaagcc catttgagat aacaacgcct gtgaaagcca ctcccgacga ggttcccctt 1561 gttgaaagcg cgactgaatc tacaaccgca aatctctcag catggtggca ggttgcgatg 1621 ggcttaatca ctaccattct tgcagcactc cttgtcttct tctggcgttg tttccttagt 1681 gcaaaaaaga ttaagttccg aaagaaagac acctttccat ggttcggcta ctcccacgga 1741 ggtcctccac cttcaccacc aggtgggcca cccccaggtt caccacggga tgcttcttac 1801 cagcaagttc cacgcactgt tgttcgtgac ctcagctttg atgaggacga tgatctacag 1861 agcgttggct tggaggaagc aggaattcgc ttcaagagtt tgatcaccac tatcgaaaga 1921 ggaaatctcc aagagcttca agcagtcatt ccagagcaca ttagtgatct caatgttctg 1981 cagtcgagcg cccatggatc tggtttctac actatggtgt cactatactt gtccacccta 2041 ggtgatgcaa taacagcttt tgagcagagg aatgatgtct caccagcaac aattcagtcg 2101 cttagaactc tcgaactcca actcgaagct cgccatctca gatttaatga agctggaact 2161 ccaacacaca tcctccagag gagcatcagc gcctctgttg ggcgagcaat tatcagatta 2221 actcaaagcg ccctccttgc atctggcgag gggtttcgaa cacgaatggc atccacgctg 2281 caacgaattg ctgatgagag atctaacaac cttacctcct tcgacgcaag agcactggac 2341 atgacgagcg agcttttcca atccatagaa gccgcccttg actctgacag tagtgatatt 2401 gtctcgctgc ttgctaatgc tgaggcctcg ctgcaggtgt acaacaactt cttcggggtt 2461 aactacgttt cgacgacgct tcttgcactt agacgcgagc taattctccg aagcgcagaa 2521 gggcgcgtcg gtgaacaacc aactggaatc tccgaagagt caaatgaaga gcttgtgcaa 2581 aaatcaatgc aaaaactaga taaagaaatc gagctcttcc aggcccagat cgacagtcag 2641 cgacgagttg cagagataac cgaatcttca aacctaaggg aaaacatcct gcaaccaata 2701 aacactgtag caaatatagc catggcagga gctttcttaa gaggtggagc acggagtcga 2761 ctcccaggtg ttctccccca agcacaaccc cacacacaag cgtttcgccc attcacgggc 2821 cgggcgcatc acctcaccac aacaggacgg gtccagcgct tcctcaggag gccgggccat 2881 tagttttccg catttgctca ttcgcacctc gcataggccg ccctactttc aacgctttct 2941 ttattacttt ggtcattaat atgatcaggc acacactcat cgtggcgccc cattgtctcc 3001 acaatagttt aaattgagca acccaaaaca ccaaaaatac catcatgcat tttgtacttc 3061 catctgcatt ccaccccatt gtgtttagct aatcgtttcg tcatcaaagt atggcgagtg 3121 taaactccac atctgtcggc ttgaccgagt gtatgggacc ttcgacaaaa ttgttacgag 3181 ttgt Transfer Plasmid 1 aaaataaaac caccacaaac aaaaccgacc aaagctatca caaacccaca cacactgctc 61 tttcatctga gcgcatagct tttctctgtc ttcacccgcg cgtgtccgtc cctcctgagc 121 cgtagccttg attccatctt gtgcttcgtt ctcctagttc tttccgcttc (Your DNA Fragments) atacct ggctctagac ttaaaccttt agagcttggg gcgaacttgg aagatagcac 3241 actacttgcg cctataaagc ccaactgtct gtgacccttc acaacgattt ctagatgatt 3301 ctacggaatt agtctttcaa atatctcggc ggcactcgac acgattgcat ccacaacaca 3361 acgcaaagtc cggtttggat atctcgaact ctagttacgg tacgtcatgg agcaacctgg 3421 cggccctacg tcatgatact tcgagctcta cctctttagc accgcgcgtt gtgccacgtc 3481 attaaacccc cagcatgttt attttctgtc gtcccgatga agctttcgag ccaagggaca 3541 cctgcataga aaacgtcact cttgtgatgc aaaaacaatg cacctgtcga tcgtcattga 3601 tcggcggctg ttgtttgaca aacccagttt ttgtccccag gaaatgtgcc Which makes this Wrong or a nonfunctional vector, see I thought that looked too easy. I wonder if the UTR's are the same, ya no they aren't that vector doesn't work, I can absolutely guarantee, after noticing that other RNA sequence didn't know it was split hold on I need a gene map for this, I realize now, which doesn't exist, which I am right is too short for that type of virus. Where okay, those were the right primers, why is it split like that. Oh, I see how this virus reproduces a Replicase and not Reverse Transcriptase...... no this virus is useless as is. Edited November 27, 2017 by Vmedvil
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 1 hour ago, Vmedvil said: Ya, I don't if it works or not the Chinese genetics team did a bad job of labeling which I am noticing is still better than the others, the LTR should be where those UTR's are but I am not certain along with it is split in RNA 1 and 2, so I dunno if it will function like that with just RNA 2. RNA 1 YMWV RNA 2 YMWV See the machinery I use is not split like that. Which makes this Wrong or a nonfunctional vector, see I thought that looked too easy. I wonder if the UTR's are the same, ya no they aren't that vector doesn't work, I can absolutely guarantee, after noticing that other RNA sequence didn't know it was split hold on I need a gene map for this, I realize now, which doesn't exist, which I am right is too short for that type of virus. Where okay, those were the right primers, why is it split like that. Oh, I see how this virus reproduces a Replicase and not Reverse Transcriptase...... no this virus is useless as is. Alright I am about to do have to do something interesting to get this to work because potyviruses have not DNA stage like Retrovirues. You know what I am gonna doing something really odd which I have never tried being way different species of virus Retrovirus/Potyvirus, So Pol, LTR3' and LTR 5' are needed for this to work and NlB removed being the stupid replicase which was the wrong type of protein, but cannot damage the protein coat of the virus which I don't like doing because I am basing the RNA structure off a Pulm Potyvirus which should be similar but not perfectly the same. So the MV-2 Vector, needs to be (Cl,Nla PRO, CP,P3,POL) Transfer Vector (LTR3'- DNA- LTR5') Now i need a protonomic and nucleotide sequence that doesn't suck for WMYV because now it is going to be integrated into DNA with HIV's machinery which there is now space for having removed 3 proteins then added 3 proteins of similar size, so WMYV will think they are its proteins and not HIV's proteins, but now pol needs to be perfectly split. Edited November 27, 2017 by Vmedvil
Vmedvil Posted November 27, 2017 Posted November 27, 2017 (edited) 2 hours ago, Vmedvil said: Alright I am about to do have to do something interesting to get this to work because potyviruses have not DNA stage like Retrovirues. You know what I am gonna doing something really odd which I have never tried being way different species of virus Retrovirus/Potyvirus, So Pol, LTR3' and LTR 5' are needed for this to work and NlB removed being the stupid replicase which was the wrong type of protein, but cannot damage the protein coat of the virus which I don't like doing because I am basing the RNA structure off a Pulm Potyvirus which should be similar but not perfectly the same. So the MV-2 Vector, needs to be (Cl,Nla PRO, CP,P3,POL) Transfer Vector (LTR3'- DNA- LTR5') Now i need a protonomic and nucleotide sequence that doesn't suck for WMYV because now it is going to be integrated into DNA with HIV's machinery which there is now space for having removed 3 proteins then added 3 proteins of similar size, so WMYV will think they are its proteins and not HIV's proteins, but now pol needs to be perfectly split. Where I am going to use this Nucleotide and Protonomic sequence for this. HIV-1 Clone Protonomic and Nucleotide sequence Pol Sequence HIV-1 Cut Perfectly ttttttaggg aagatctggc cttcccacaa gggaaggcca gggaattttc 1681 ttcagagcag accagagcca acagccccac cagaagagag cttcaggttt ggggaagaga 1741 caacaactcc ctctcagaag caggagccga tagacaagga actgtatcct ttagcttccc 1801 tcagatcact ctttggcagc gacccctcgt cacaataaag ataggggggc aattaaagga 1861 agctctatta gatacaggag cagatgatac agtattagaa gaaatgaatt tgccaggaag 1921 atggaaacca aaaatgatag ggggaattgg aggttttatc aaagtaagac agtatgatca 1981 gatactcata gaaatctgcg gacataaagc tataggtaca gtattagtag gacctacacc 2041 tgtcaacata attggaagaa atctgttgac tcagattggc tgcactttaa attttcccat 2101 tagtcctatt gagactgtac cagtaaaatt aaagccagga atggatggcc caaaagttaa 2161 acaatggcca ttgacagaag aaaaaataaa agcattagta gaaatttgta cagaaatgga 2221 aaaggaagga aaaatttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc 2281 cataaagaaa aaagacagta ctaaatggag aaaattagta gatttcagag aacttaataa 2341 gagaactcaa gatttctggg aagttcaatt aggaatacca catcctgcag ggttaaaaca 2401 gaaaaaatca gtaacagtac tggatgtggg cgatgcatat ttttcagttc ccttagataa 2461 agacttcagg aagtatactg catttaccat acctagtata aacaatgaga caccagggat 2521 tagatatcag tacaatgtgc ttccacaggg atggaaagga tcaccagcaa tattccagtg 2581 tagcatgaca aaaatcttag agccttttag aaaacaaaat ccagacatag tcatctatca 2641 atacatggat gatttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat 2701 agaggaactg agacaacatc tgttgaggtg gggatttacc acaccagaca aaaaacatca 2761 gaaagaacct ccattccttt ggatgggtta tgaactccat cctgataaat ggacagtaca 2821 gcctatagtg ctgccagaaa aggacagctg gactgtcaat gacatacaga aattagtggg 2881 aaaattgaat tgggcaagtc agatttatgc agggattaaa gtaaggcaat tatgtaaact 2941 tcttagggga accaaagcac taacagaagt agtaccacta acagaagaag cagagctaga 3001 actggcagaa aacagggaga ttctaaaaga accggtacat ggagtgtatt atgacccatc 3061 aaaagactta atagcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta 3121 tcaagagcca tttaaaaatc tgaaaacagg aaagtatgca agaatgaagg gtgcccacac 3181 taatgatgtg aaacaattaa cagaggcagt acaaaaaata gccacagaaa gcatagtaat 3241 atggggaaag actcctaaat ttaaattacc catacaaaag gaaacatggg aagcatggtg 3301 gacagagtat tggcaagcca cctggattcc tgagtgggag tttgtcaata cccctccctt 3361 agtgaagtta tggtaccagt tagagaaaga acccataata ggagcagaaa ctttctatgt 3421 agatggggca gccaataggg aaactaaatt aggaaaagca ggatatgtaa ctgacagagg 3481 aagacaaaaa gttgtccccc taacggacac aacaaatcag aagactgagt tacaagcaat 3541 tcatctagct ttgcaggatt cgggattaga agtaaacata gtgacagact cacaatatgc 3601 attgggaatc attcaagcac aaccagataa gagtgaatca gagttagtca gtcaaataat 3661 agagcagtta ataaaaaagg aaaaagtcta cctggcatgg gtaccagcac acaaaggaat 3721 tggaggaaat gaacaagtag ataaattggt cagtgctgga atcaggaaag tactattttt 3781 agatggaata gataaggccc aagaagaaca tgagaaatat cacagtaatt ggagagcaat 3841 ggctagtgat tttaacctac cacctgtagt agcaaaagaa atagtagcca gctgtgataa 3901 atgtcagcta aaaggggaag ccatgcatgg acaagtagac tgtagcccag gaatatggca 3961 gctagattgt acacatttag aaggaaaagt tatcttggta gcagttcatg tagccagtgg 4021 atatatagaa gcagaagtaa ttccagcaga gacagggcaa gaaacagcat acttcctctt 4081 aaaattagca ggaagatggc cagtaaaaac agtacataca gacaatggca gcaatttcac 4141 cagtactaca gttaaggccg cctgttggtg ggcggggatc aagcaggaat ttggcattcc 4201 ctacaatccc caaagtcaag gagtaataga atctatgaat aaagaattaa agaaaattat 4261 aggacaggta agagatcagg ctgaacatct taagacagca gtacaaatgg cagtattcat 4321 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 4381 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 4441 ttttcgggtt tattacaggg acagcagaga tccagtttgg aaaggaccag caaagctcct 4501 ctggaaaggt gaaggggcag tagtaataca agataatagt gacataaaag tagtgccaag 4561 aagaaaagca aagatcatca gggattatgg aaaacagatg gcaggtgatg attgtgtggc 4621 aagtagacag gatgaggatt aa Now for to Edit RNA 1 of that Wheat Virus RNA 1 Wheat Virus Protonomic and Nucleotide sequence Cl,Nla PRO, CP,P3 Sequence Removed replicase Perfectly(Nlb) tggagca gacagcagcc 181 cagaccgccg cgcgccacgc cacacacacg aactccgcac aagagcagcc tcctgtcagc 241 cctatgaatc tgtctgcaaa ctttgttgcc cctgagcttt tctacgcttc aaatgtcgag 301 aagatcaaga gtatcatgag gatgcgtagc acgacgcgca tcattgatgc gatatccagg 361 gatttcgagc tcgttgcttt tctcatactc agccccgcac atctcatgca gctagaaacc 421 acactacgct gcgagatgaa atccagcatt gtacctattg caaactcaga tgctggtttt 481 gagacttgcg ccgtcctcaa aacagcgctc gatggaatga agtatcactt tggaacaacc 541 acactcgagc aaggctggac atccatgatg aaacacgctg acagctgcct gcaagagagt 601 tcttcctctg ccgtcgccat gttacaaact caagttcaga aagtcggctc cctcttaatc 661 agtggaaaaa atcgcgtaga aagttgcgaa ctttatgtct tacacctcac cgcgcgcgct 721 ttcaggaccg aatatggatt aaagggtact tgctttggag aacactgcgc tttgttgcac 781 gacttaaaag catgcatttg tggtaccgtg ccaaaagagt ttttgtgggc gaaaaccaaa 841 aagcactcca tgttcactat cccagagtgg atcgcacgta cgccaacaga ctgtttcatg 901 ctgtgtcttc gcgctatacc tatctttcgt aggtgcacga tggcaatgtc tttactttat 961 tggtcgtgtg tagcaactgt taacctccct tctgttatgg cttttctttt taagcgtcag 1021 ttcactaaat acatagccca ttccttcgcc aagcactcca tctatttcct tatgttaggt 1081 attgtagccc tactatgggc attccgcact tatgcttcga aaaacccaaa aatagctctc 1141 caagctagga gcgcaaacga aaaggaaaag aagctgatga tgttacttgc aagcgccgct 1201 ggaatcacat atctatttga ctacgacatc gcagaagccc tagggaattc tctccataaa 1261 ataagtcgct tgtcgtctta tctaatggat gatcatcaag gaatagcctc tcgcatgttt 1321 agcgctagct acggacttca agctgggaac aatgtggaag atgcagtcac cacaatcatt 1381 agtgatctct tgtctgtcac ctttcaaatt gtcgacgaag atgcaactgc tggagcttta 1441 gaagaggtca gcgacacaac tttcagtagt tgggttagcg ttaacactct cgctggaagg 1501 aacatgtcac gccccctgca ataccctgtg aatcagactt acccgctcac accaatgaac 1561 gttttgctgc aagctcaagc tatggtcgat tcggacaact gttggtcaat ggtcgttgga 1621 cacacgggat ccggaaaatc cacgtatctc cctgtccagt actctaacta ccttgccaag 1681 aaacctgaac gtcgccagca aattctagtt tgcgagccca cgcaagcagc cacagaaaat 1741 gtctgctcag gaatcgctgc caacttaggg agagctgtat atggaagaca tgaaggttgg 1801 tcgcgcatgg gagatcactg cattcaagtt atgacctatg gatcagctct acagtgccac 1861 gcaatggatc caggcttcat atcaaccttt gacgcaatat tccttgatga agctcacgat 1921 gtcaaagagc attcacttgt cttcgaaagc atctgcaaca acttcgagtc tgttaggaag 1981 ttctacgtgt cagcaacccc ccgagatggc agttcatgcc cggatgcaac acgcaaattc 2041 cctctaacag ttcagcagag tgtatgcgat tcatataaga aattcatagc cgcacagggt 2101 ggacacgatc ttctcgacgt cactaaacac gacaccgttc ttgtttttct cgctggaaga 2161 ccagaatgcg tcaaagctgc aaatgcatgg aatgctaaca tcactggaga aaagcgagct 2221 ttctcgctat caagcgacaa cttcaccacc gattttgcaa tgctaacgga gcgcctcaaa 2281 acgcacaaga ccatcatctt cacaacgaac atcatcgaaa ctggtgttac ccttagcgtt 2341 gactgtgttg ttgactttgg ctataccatg cgaccctccc tcgatctaaa ccacaaaaca 2401 ctacgccttg aacgccgtag agtaactgca aatgagagaa agcaaaggat cggtagagct 2461 ggtcgcctta aggaaggata tgccattgtg tgtggcgatg aagatcgatc tatcaatgtc 2521 gtctccccgg atgttctgta tggtgccgcg cttcttagct tcaagcacaa tgtacccttc 2581 tacatgaacg acacctttga aagctcgtgg cttgaaggag ttactaagtc tcaggctgac 2641 actatgacaa ttttcaagct ccccatattc ctaacgcgcg atcttgttaa ctccgacggc 2701 tctgtagcaa aggagtttct tgacattctg aagaagcacc agttcacaac gagcgacatc 2761 aagcaagcgc cacacagcat agctcgtcac ctctttccaa cttgggcctc atactttgca 2821 ctcttccaat caatcaacta cggagaggag aaagacgaga tccccagcaa ccttcgccat 2881 gcgcgagttc ccttctccat ttcaacgctg tctaaattcg actggaatgc cttggcgctt 2941 gcatgtgagc agcatcgccc aacagtggca agctcattcc aaagccttga tcagccagcc 3001 cgcgttgtca ccctacaaac aaactctgca aacatcctcg gaagcatcgc ccatctctgc 3061 aacatgcgta acaattacaa aaccttgatt gacagcaaca accatgtaaa gcaatcaatg 3121 gcaaccaacg tcatgttcaa gtggttctca tcatcccaca tcacagctaa tcttgacaga 3181 aatttatccc gatgcttagc caacttgtca gttgtggaaa ccacgattag cgcgctaaag 3241 cagatttcag ctgaaaactc gcaggtgatg gcttctccaa cacttcagag tcacctagag 3301 aacataatcg aactacaatc aaacgatatg ttaactgatg aaacactatc aaacgctcta 3361 ggcattttca acccaaagac aaacctcttc ctccttctcg caaccaaagg tttcaaactt 3421 gtttacgtta tctgcctgct catattaata aacctcatct accgcttgct cttccactgg 3481 agagcttggt tgaagaacaa gaacgataac gtggaccccg atgccctcac caacaccatg 3541 acagtgcagg aaggaagcga aattctcaag gaagtattga agatgactcc agcgatgcgc 3601 agggaggtga ccaaggatat gaaagttgca gtcgctgata acgacagcac attctccttc 3661 gtgttccctt acgaacacat cgacctcgag ggaaagggaa acaagtatcg accacgagag 3721 gacgcacgtc tgatgtactc cacgagagac gacgcaacct tcgatacatg gaacgagaaa 3781 gctaaggaga aaaggaagaa gatatcagac cgcgcagagc cagagatgag acaaccttac 3841 cagaagcgtc catactataa cttctacgac cttcaaactg atagcaacat tctcgaggca 3901 atattctaca ccacggaagg tgacgaattc ttccggacgg caaatcccaa caaggacatg 3961 cactcagtag ctgaaaaact aaaagccttc cttgacacaa aaccaatcat aggcagacac 4021 cagcgccagc tccttgggga aaccgctcag gtcgttatca aagacgacaa aggcactgcc 4081 cacagaatgg acatatcaac ccacaacccc aacactctta agaacaatgg atcaggaaga 4141 gttggatatg atgagcatcg cggcgatttc aggcaagaga gccctgcact tgagtcaccg 4201 tacgagttgg aagctgaatt cggaaactca aaggatggag tcatgcttga agcatcaaca 4261 ggaattctcc tatcccaggt cagcgttgac gttgcgactc gaattggaag aataagcatc 4321 ggaacgttca atgtcaattg cttcctctac agtgattgga tactagcacc tgctcatctt 4381 caggatcgaa acggcgccgt caccatcgaa ttcccagacc aaacggtgag taccacaacc 4441 gacaatctaa atgcgcacgg tgttaagcga ttttacggac ttgacctcat agccattcga 4501 agaccaccaa gcctccgacc tgagaaaaag ctcgttaaag cgttcgccat cacagagcca 4561 gtgatagcac agatggtctt cattgacagc caagggatcc gaaagttttc tcaatccgac 4621 tggatcagac aggaagttgg ttctaacaga tggtcacaca agatttcaac tcaaagtggg 4681 atgtgcggat gccctgtttt ggatgttgga aagaaccgtc tcattggaat tcacgttgcc 4741 acgaaccaca ccacaggacg aaatgaattc cagcctttct cacgcgaggc tgttgacttc 4801 atcaacgggc caggaaacaa ggttcctttc agcccttgga ccttctgccg ccccgcctgt 4861 ggatacaagc aaggagacgc caccttcgct tcacgcgcca tacagaccca agcctccgac 4921 attcttggca agatcaacaa ctccatcctt ggctttggaa gtgatttgaa aggccagctt 4981 gttcagcccg taacaccagc tctacgcact cgctttgaag ccctgttcgg tggtggaagc 5041 ttcgaactcg tcggaacaat gaacaaagga ctcatagaca agcacacaat tgttggggag 5101 aacgacaacg ttcatgattt catgagagaa caccccactt ttgcttggct tcagggtttc 5161 atggacgaat acgcaccaag tgtcttgaac tactcggctt actacaagga cctttgcaag 5221 tacaacagga agaagcacca actctccttc aacccccacg agcttcggag tgccaccgct 5281 ggaatgatca gaatgttgga agacgccggc ctaactcaag gcgacgtgag gacaccccaa 5341 caggttgttt cagacatgca gtggaacact tccgccggac caagctacca gggcaagaaa 5401 agagacgttt gcgcacatct cagcgagcag gaagttctac accttgcaga aacgtcgcga 5461 cagcagtttc tagcctgcaa ttccatcggc atttggaacg gatcactcaa agcagagctt 5521 aggacgattg agaaagtaga agctgagaaa accagagttt tcacggcttc gccaatcacg 5581 agtctattcg ccatgaagtt ctacgttgac gatttcaaca agaagttcta tgcgacaaac 5641 ctgaaagctc cacacacggt gggaattaac aaattcagca gaggctggga aatgctccac 5701 gacaagctca accgcccagg ttggctgcac ggcagtggcg atgggtcacg atttgatagc 5761 tcgattgacc ctttcttctt cgacataatc aaggagattc gaaagcactt tctaccagtt 5821 gagcaccatc gcgcaatcga cctaatatac gacgagattc tcaacaccaa catttgtttg 5881 gcaaatggca tggtgattcg aaagaacgtt gggaataaca gtgggcagcc aagcacggtc 5941 gtagacaaca cactcgttct catggtctct tttctctacg cttacattca taaaaccggg 6001 gaccacatgc ccaagaaact caacgataga tttgttttcg tctgcaatgg tgacgacaac 6061 aaatttgcca tttccccaga attcgacgcc gagttcggtc atgacttttc accggagctc 6121 acagagctag gtctgacgta cgagtttgac gacataacag atgacatctg tgcaaacccc 6181 tacatgtccc tgaccatggt ccgcactccc ttcggaattg ggttctcttt gcccgtggaa 6241 cgcattgtag caataatgca gtgggctaag aagggcggcg ttctgcactc ttacttggca 6301 ggaatctcag ccatttacga aagcttcaat acaccaaagt tgttcaaatc agtctatgcg 6361 tatttgttat ggctgacaga ggaacacggc tctgatatcc tagctgcaat gaccggaaca 6421 gcgacagctc tcccaattcc ctccatgctc gatgtgtacc gcctccacta tggtaatagc 6481 agcattgagc tccaag taaacc 7381 atcacccccc tccgcctttt atatttgcac tttaaatttt cgcaaaccat caccaccttt 7441 cacggctacg gttacgatct ttaatttttg cacttgtatc aggctcggga cggttctatg 7501 cacaatatgt tttcgacagt tgccaaacca gtattttcca tgaggaagag tatggcgagg 7561 cttgagcagc cgtttttaat catacgtcgt tgtatggtgg cgcattcagt gcgtttacga 7621 gtaccagaag gtaat Recombined Retroviral Wheat Mosiac Vector (MV-2) tggagca gacagcagcc 181 cagaccgccg cgcgccacgc cacacacacg aactccgcac aagagcagcc tcctgtcagc 241 cctatgaatc tgtctgcaaa ctttgttgcc cctgagcttt tctacgcttc aaatgtcgag 301 aagatcaaga gtatcatgag gatgcgtagc acgacgcgca tcattgatgc gatatccagg 361 gatttcgagc tcgttgcttt tctcatactc agccccgcac atctcatgca gctagaaacc 421 acactacgct gcgagatgaa atccagcatt gtacctattg caaactcaga tgctggtttt 481 gagacttgcg ccgtcctcaa aacagcgctc gatggaatga agtatcactt tggaacaacc 541 acactcgagc aaggctggac atccatgatg aaacacgctg acagctgcct gcaagagagt 601 tcttcctctg ccgtcgccat gttacaaact caagttcaga aagtcggctc cctcttaatc 661 agtggaaaaa atcgcgtaga aagttgcgaa ctttatgtct tacacctcac cgcgcgcgct 721 ttcaggaccg aatatggatt aaagggtact tgctttggag aacactgcgc tttgttgcac 781 gacttaaaag catgcatttg tggtaccgtg ccaaaagagt ttttgtgggc gaaaaccaaa 841 aagcactcca tgttcactat cccagagtgg atcgcacgta cgccaacaga ctgtttcatg 901 ctgtgtcttc gcgctatacc tatctttcgt aggtgcacga tggcaatgtc tttactttat 961 tggtcgtgtg tagcaactgt taacctccct tctgttatgg cttttctttt taagcgtcag 1021 ttcactaaat acatagccca ttccttcgcc aagcactcca tctatttcct tatgttaggt 1081 attgtagccc tactatgggc attccgcact tatgcttcga aaaacccaaa aatagctctc 1141 caagctagga gcgcaaacga aaaggaaaag aagctgatga tgttacttgc aagcgccgct 1201 ggaatcacat atctatttga ctacgacatc gcagaagccc tagggaattc tctccataaa 1261 ataagtcgct tgtcgtctta tctaatggat gatcatcaag gaatagcctc tcgcatgttt 1321 agcgctagct acggacttca agctgggaac aatgtggaag atgcagtcac cacaatcatt 1381 agtgatctct tgtctgtcac ctttcaaatt gtcgacgaag atgcaactgc tggagcttta 1441 gaagaggtca gcgacacaac tttcagtagt tgggttagcg ttaacactct cgctggaagg 1501 aacatgtcac gccccctgca ataccctgtg aatcagactt acccgctcac accaatgaac 1561 gttttgctgc aagctcaagc tatggtcgat tcggacaact gttggtcaat ggtcgttgga 1621 cacacgggat ccggaaaatc cacgtatctc cctgtccagt actctaacta ccttgccaag 1681 aaacctgaac gtcgccagca aattctagtt tgcgagccca cgcaagcagc cacagaaaat 1741 gtctgctcag gaatcgctgc caacttaggg agagctgtat atggaagaca tgaaggttgg 1801 tcgcgcatgg gagatcactg cattcaagtt atgacctatg gatcagctct acagtgccac 1861 gcaatggatc caggcttcat atcaaccttt gacgcaatat tccttgatga agctcacgat 1921 gtcaaagagc attcacttgt cttcgaaagc atctgcaaca acttcgagtc tgttaggaag 1981 ttctacgtgt cagcaacccc ccgagatggc agttcatgcc cggatgcaac acgcaaattc 2041 cctctaacag ttcagcagag tgtatgcgat tcatataaga aattcatagc cgcacagggt 2101 ggacacgatc ttctcgacgt cactaaacac gacaccgttc ttgtttttct cgctggaaga 2161 ccagaatgcg tcaaagctgc aaatgcatgg aatgctaaca tcactggaga aaagcgagct 2221 ttctcgctat caagcgacaa cttcaccacc gattttgcaa tgctaacgga gcgcctcaaa 2281 acgcacaaga ccatcatctt cacaacgaac atcatcgaaa ctggtgttac ccttagcgtt 2341 gactgtgttg ttgactttgg ctataccatg cgaccctccc tcgatctaaa ccacaaaaca 2401 ctacgccttg aacgccgtag agtaactgca aatgagagaa agcaaaggat cggtagagct 2461 ggtcgcctta aggaaggata tgccattgtg tgtggcgatg aagatcgatc tatcaatgtc 2521 gtctccccgg atgttctgta tggtgccgcg cttcttagct tcaagcacaa tgtacccttc 2581 tacatgaacg acacctttga aagctcgtgg cttgaaggag ttactaagtc tcaggctgac 2641 actatgacaa ttttcaagct ccccatattc ctaacgcgcg atcttgttaa ctccgacggc 2701 tctgtagcaa aggagtttct tgacattctg aagaagcacc agttcacaac gagcgacatc 2761 aagcaagcgc cacacagcat agctcgtcac ctctttccaa cttgggcctc atactttgca 2821 ctcttccaat caatcaacta cggagaggag aaagacgaga tccccagcaa ccttcgccat 2881 gcgcgagttc ccttctccat ttcaacgctg tctaaattcg actggaatgc cttggcgctt 2941 gcatgtgagc agcatcgccc aacagtggca agctcattcc aaagccttga tcagccagcc 3001 cgcgttgtca ccctacaaac aaactctgca aacatcctcg gaagcatcgc ccatctctgc 3061 aacatgcgta acaattacaa aaccttgatt gacagcaaca accatgtaaa gcaatcaatg 3121 gcaaccaacg tcatgttcaa gtggttctca tcatcccaca tcacagctaa tcttgacaga 3181 aatttatccc gatgcttagc caacttgtca gttgtggaaa ccacgattag cgcgctaaag 3241 cagatttcag ctgaaaactc gcaggtgatg gcttctccaa cacttcagag tcacctagag 3301 aacataatcg aactacaatc aaacgatatg ttaactgatg aaacactatc aaacgctcta 3361 ggcattttca acccaaagac aaacctcttc ctccttctcg caaccaaagg tttcaaactt 3421 gtttacgtta tctgcctgct catattaata aacctcatct accgcttgct cttccactgg 3481 agagcttggt tgaagaacaa gaacgataac gtggaccccg atgccctcac caacaccatg 3541 acagtgcagg aaggaagcga aattctcaag gaagtattga agatgactcc agcgatgcgc 3601 agggaggtga ccaaggatat gaaagttgca gtcgctgata acgacagcac attctccttc 3661 gtgttccctt acgaacacat cgacctcgag ggaaagggaa acaagtatcg accacgagag 3721 gacgcacgtc tgatgtactc cacgagagac gacgcaacct tcgatacatg gaacgagaaa 3781 gctaaggaga aaaggaagaa gatatcagac cgcgcagagc cagagatgag acaaccttac 3841 cagaagcgtc catactataa cttctacgac cttcaaactg atagcaacat tctcgaggca 3901 atattctaca ccacggaagg tgacgaattc ttccggacgg caaatcccaa caaggacatg 3961 cactcagtag ctgaaaaact aaaagccttc cttgacacaa aaccaatcat aggcagacac 4021 cagcgccagc tccttgggga aaccgctcag gtcgttatca aagacgacaa aggcactgcc 4081 cacagaatgg acatatcaac ccacaacccc aacactctta agaacaatgg atcaggaaga 4141 gttggatatg atgagcatcg cggcgatttc aggcaagaga gccctgcact tgagtcaccg 4201 tacgagttgg aagctgaatt cggaaactca aaggatggag tcatgcttga agcatcaaca 4261 ggaattctcc tatcccaggt cagcgttgac gttgcgactc gaattggaag aataagcatc 4321 ggaacgttca atgtcaattg cttcctctac agtgattgga tactagcacc tgctcatctt 4381 caggatcgaa acggcgccgt caccatcgaa ttcccagacc aaacggtgag taccacaacc 4441 gacaatctaa atgcgcacgg tgttaagcga ttttacggac ttgacctcat agccattcga 4501 agaccaccaa gcctccgacc tgagaaaaag ctcgttaaag cgttcgccat cacagagcca 4561 gtgatagcac agatggtctt cattgacagc caagggatcc gaaagttttc tcaatccgac 4621 tggatcagac aggaagttgg ttctaacaga tggtcacaca agatttcaac tcaaagtggg 4681 atgtgcggat gccctgtttt ggatgttgga aagaaccgtc tcattggaat tcacgttgcc 4741 acgaaccaca ccacaggacg aaatgaattc cagcctttct cacgcgaggc tgttgacttc 4801 atcaacgggc caggaaacaa ggttcctttc agcccttgga ccttctgccg ccccgcctgt 4861 ggatacaagc aaggagacgc caccttcgct tcacgcgcca tacagaccca agcctccgac 4921 attcttggca agatcaacaa ctccatcctt ggctttggaa gtgatttgaa aggccagctt 4981 gttcagcccg taacaccagc tctacgcact cgctttgaag ccctgttcgg tggtggaagc 5041 ttcgaactcg tcggaacaat gaacaaagga ctcatagaca agcacacaat tgttggggag 5101 aacgacaacg ttcatgattt catgagagaa caccccactt ttgcttggct tcagggtttc 5161 atggacgaat acgcaccaag tgtcttgaac tactcggctt actacaagga cctttgcaag 5221 tacaacagga agaagcacca actctccttc aacccccacg agcttcggag tgccaccgct 5281 ggaatgatca gaatgttgga agacgccggc ctaactcaag gcgacgtgag gacaccccaa 5341 caggttgttt cagacatgca gtggaacact tccgccggac caagctacca gggcaagaaa 5401 agagacgttt gcgcacatct cagcgagcag gaagttctac accttgcaga aacgtcgcga 5461 cagcagtttc tagcctgcaa ttccatcggc atttggaacg gatcactcaa agcagagctt 5521 aggacgattg agaaagtaga agctgagaaa accagagttt tcacggcttc gccaatcacg 5581 agtctattcg ccatgaagtt ctacgttgac gatttcaaca agaagttcta tgcgacaaac 5641 ctgaaagctc cacacacggt gggaattaac aaattcagca gaggctggga aatgctccac 5701 gacaagctca accgcccagg ttggctgcac ggcagtggcg atgggtcacg atttgatagc 5761 tcgattgacc ctttcttctt cgacataatc aaggagattc gaaagcactt tctaccagtt 5821 gagcaccatc gcgcaatcga cctaatatac gacgagattc tcaacaccaa catttgtttg 5881 gcaaatggca tggtgattcg aaagaacgtt gggaataaca gtgggcagcc aagcacggtc 5941 gtagacaaca cactcgttct catggtctct tttctctacg cttacattca taaaaccggg 6001 gaccacatgc ccaagaaact caacgataga tttgttttcg tctgcaatgg tgacgacaac 6061 aaatttgcca tttccccaga attcgacgcc gagttcggtc atgacttttc accggagctc 6121 acagagctag gtctgacgta cgagtttgac gacataacag atgacatctg tgcaaacccc 6181 tacatgtccc tgaccatggt ccgcactccc ttcggaattg ggttctcttt gcccgtggaa 6241 cgcattgtag caataatgca gtgggctaag aagggcggcg ttctgcactc ttacttggca 6301 ggaatctcag ccatttacga aagcttcaat acaccaaagt tgttcaaatc agtctatgcg 6361 tatttgttat ggctgacaga ggaacacggc tctgatatcc tagctgcaat gaccggaaca 6421 gcgacagctc tcccaattcc ctccatgctc gatgtgtacc gcctccacta tggtaatagc 6481 agcattgagc tccaag ttttttaggg aagatctggc cttcccacaa gggaaggcca gggaattttc 1681 ttcagagcag accagagcca acagccccac cagaagagag cttcaggttt ggggaagaga 1741 caacaactcc ctctcagaag caggagccga tagacaagga actgtatcct ttagcttccc 1801 tcagatcact ctttggcagc gacccctcgt cacaataaag ataggggggc aattaaagga 1861 agctctatta gatacaggag cagatgatac agtattagaa gaaatgaatt tgccaggaag 1921 atggaaacca aaaatgatag ggggaattgg aggttttatc aaagtaagac agtatgatca 1981 gatactcata gaaatctgcg gacataaagc tataggtaca gtattagtag gacctacacc 2041 tgtcaacata attggaagaa atctgttgac tcagattggc tgcactttaa attttcccat 2101 tagtcctatt gagactgtac cagtaaaatt aaagccagga atggatggcc caaaagttaa 2161 acaatggcca ttgacagaag aaaaaataaa agcattagta gaaatttgta cagaaatgga 2221 aaaggaagga aaaatttcaa aaattgggcc tgaaaatcca tacaatactc cagtatttgc 2281 cataaagaaa aaagacagta ctaaatggag aaaattagta gatttcagag aacttaataa 2341 gagaactcaa gatttctggg aagttcaatt aggaatacca catcctgcag ggttaaaaca 2401 gaaaaaatca gtaacagtac tggatgtggg cgatgcatat ttttcagttc ccttagataa 2461 agacttcagg aagtatactg catttaccat acctagtata aacaatgaga caccagggat 2521 tagatatcag tacaatgtgc ttccacaggg atggaaagga tcaccagcaa tattccagtg 2581 tagcatgaca aaaatcttag agccttttag aaaacaaaat ccagacatag tcatctatca 2641 atacatggat gatttgtatg taggatctga cttagaaata gggcagcata gaacaaaaat 2701 agaggaactg agacaacatc tgttgaggtg gggatttacc acaccagaca aaaaacatca 2761 gaaagaacct ccattccttt ggatgggtta tgaactccat cctgataaat ggacagtaca 2821 gcctatagtg ctgccagaaa aggacagctg gactgtcaat gacatacaga aattagtggg 2881 aaaattgaat tgggcaagtc agatttatgc agggattaaa gtaaggcaat tatgtaaact 2941 tcttagggga accaaagcac taacagaagt agtaccacta acagaagaag cagagctaga 3001 actggcagaa aacagggaga ttctaaaaga accggtacat ggagtgtatt atgacccatc 3061 aaaagactta atagcagaaa tacagaagca ggggcaaggc caatggacat atcaaattta 3121 tcaagagcca tttaaaaatc tgaaaacagg aaagtatgca agaatgaagg gtgcccacac 3181 taatgatgtg aaacaattaa cagaggcagt acaaaaaata gccacagaaa gcatagtaat 3241 atggggaaag actcctaaat ttaaattacc catacaaaag gaaacatggg aagcatggtg 3301 gacagagtat tggcaagcca cctggattcc tgagtgggag tttgtcaata cccctccctt 3361 agtgaagtta tggtaccagt tagagaaaga acccataata ggagcagaaa ctttctatgt 3421 agatggggca gccaataggg aaactaaatt aggaaaagca ggatatgtaa ctgacagagg 3481 aagacaaaaa gttgtccccc taacggacac aacaaatcag aagactgagt tacaagcaat 3541 tcatctagct ttgcaggatt cgggattaga agtaaacata gtgacagact cacaatatgc 3601 attgggaatc attcaagcac aaccagataa gagtgaatca gagttagtca gtcaaataat 3661 agagcagtta ataaaaaagg aaaaagtcta cctggcatgg gtaccagcac acaaaggaat 3721 tggaggaaat gaacaagtag ataaattggt cagtgctgga atcaggaaag tactattttt 3781 agatggaata gataaggccc aagaagaaca tgagaaatat cacagtaatt ggagagcaat 3841 ggctagtgat tttaacctac cacctgtagt agcaaaagaa atagtagcca gctgtgataa 3901 atgtcagcta aaaggggaag ccatgcatgg acaagtagac tgtagcccag gaatatggca 3961 gctagattgt acacatttag aaggaaaagt tatcttggta gcagttcatg tagccagtgg 4021 atatatagaa gcagaagtaa ttccagcaga gacagggcaa gaaacagcat acttcctctt 4081 aaaattagca ggaagatggc cagtaaaaac agtacataca gacaatggca gcaatttcac 4141 cagtactaca gttaaggccg cctgttggtg ggcggggatc aagcaggaat ttggcattcc 4201 ctacaatccc caaagtcaag gagtaataga atctatgaat aaagaattaa agaaaattat 4261 aggacaggta agagatcagg ctgaacatct taagacagca gtacaaatgg cagtattcat 4321 ccacaatttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga 4381 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa 4441 ttttcgggtt tattacaggg acagcagaga tccagtttgg aaaggaccag caaagctcct 4501 ctggaaaggt gaaggggcag tagtaataca agataatagt gacataaaag tagtgccaag 4561 aagaaaagca aagatcatca gggattatgg aaaacagatg gcaggtgatg attgtgtggc 4621 aagtagacag gatgaggatt aa taaacc 7381 atcacccccc tccgcctttt atatttgcac tttaaatttt cgcaaaccat caccaccttt 7441 cacggctacg gttacgatct ttaatttttg cacttgtatc aggctcggga cggttctatg 7501 cacaatatgt tttcgacagt tgccaaacca gtattttcca tgaggaagag tatggcgagg 7561 cttgagcagc cgtttttaat catacgtcgt tgtatggtgg cgcattcagt gcgtttacga 7621 gtaccagaag gtaat Transfer Plasmid LTR3' - DNA - LTR5' 1 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 61 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 121 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca (Your DNA Fragments) gg aagggctaat 8641 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt 8701 ccctgattgg cagaactaca caccaggacc agggatcaga tatccactga cctttggatg 8761 gcgctacaag ctagtaccag ttgagccaga gaagttagaa gaagccaaca aaggagagaa 8821 caccagcttg ttacaccctg tgagcctgca tggaatggat gacccggaga gagaagtgtt 8881 agagtggagg tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga 8941 gtacttcaag aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc 9001 agggaggcgt ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc 9061 agctgctttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct 9121 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag 9181 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt 9241 cagtgtggaa aatctctagc agt Now it is fixed that should work as I thought the original one would, there you go now it insert's its RNA into the host's DNA instead of being mRNA., Viral Machinery HIV-1 the rest Wheat Yellow Mosaic Virus minus Nlb, non reproducing in this form don't screw with its code still stands. Edited November 27, 2017 by Vmedvil
PapainLCP Posted December 9, 2017 Posted December 9, 2017 How did you detect your gRNAs? Are you also using biolistics to avoid GMO regulation?
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