Ferramenta para desenhar os primers

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Jalview e Est2Genome
Ferramenta para desenhar os primers;
Ferramenta para identificar onde estão os íntrons;
Refazer com o segundo e terceiro gene do contig um;
Sequência da versão genômica desse gene é comparada com a do RNAm (versão cDNA);
Contig 1
1 – Blastar o contig 1 blastx – Para chegar na versão cDNA do gene;
>Contig1 
TTCTTCACTTGGTTTTTATCACTATATCAGACTACTACTGATAAAATAAATGGAATGTACCGGTTAATAGATCAGGTTTTCTTTTCTTTTTTTTTGGAATTTCTTTGTCTCCTCAAATTGTATTAATTTTCAGTTTGTATGCTTCTTTTTCTGAAATTGAAATTTTATTATTATAATGGATGAAAATTGAAATTTTTGAGAGTGGCCGAATTTTTTAATTTCCGTGGCCATGCTGTGAAACTAAGTTTTTTGTTGTAGATCTCAAAAAAAATCCTGGGAGGTGTCTGTCGCTCTAACTTATTTATGTGTGCAAAATCTGAAGAACATTTTGAAGAACGAGTACGGTCAATGGGGAGTGCTATCCAGAATAATTAAAATATCGGAAAAGGCTGAAAAAATCGTGCGATTTTTCACCCCGCTTTAGAACCAATCGACTAAAATGTGCAAAGTCTGGTCTTGTAGCTCTCATTTTGGAACAACTTAAAAACATGTCTTGTACCTCGTGATTCTTATATTCTCTAGAAACCATATTAGAAATGTTCGACGTATAACTTTTCTAAGAGTAAAATTATCGACAATTCACAACTTGGATGCTCCCTAGCAAAATATGATGAAGCTCTTTGCAGTTGATATAGTCTACTACAATTTTCATGAAATCTGCAATTATCTCCAAACTGTTCGTTCTATAAAATTGTTAGAACTGTCTTGCAAACAACTTTGGTCCACCTTGGAAAATCAAACGATCCTAACAGAACCATTTCTCTAAAATCTACTTATTAATATATCAACTCTGTATTATCATCATTCACTTATCAAGAGATACACTGGGAGATTGAGGCAACTATAGAATAGAATACAACTGTATGTTGTAGTTCTTTATTCTTGCGTCCTTCTTACAAGTTATCATTCTCTCTCTTTTATTCTTTCATTAACCTTTTTTGTCGGCCGGTTCTTATCGATTGTCTAACTTCATTTATTTTCTATAATCGTTGAAGAACTGTTATTAAGATTTCAATTTCTACAAATTATTATCTGCTCTTATCAGTTGTTGCCACTACATACCTTTCTTTATTTATTTAATAAATTTCCAGCCAAACCATGAAATCTTGGCTTCTATTGCTTGGAACAATAGGTTGGTAACCGTGGAGATGTTCTTTTCATTCCAAGTACATTATATCCAGTCCATGCCCAGACAATGTCAGTTCTTGCAAGAGAGTTGGCTGAACGAGGACACAAAGTGACATGGCTGGAAATTGGCCCAAAAGGGTAATAATCAGGATAACTATTTAGATGAGAACATTATAGAAGTTTGGCGTAGATATTGGAAAAAAATTGATTCTATTAACTTGGGTTATATGAAATTAAAAAAAGATTAGTTTTGGCAGATACCAAATTTTGAAAAATGTGGACTTTTTTGACCGAATGCCGAATAATTTATATTTTATTTTTGAAAAGTTTCACTTTATTTTTTTGGTGATTTCAGCACCCTAACTAGACCATAAGAGCAACATGCTCACCTTCTTCGTTATTTTTTTTTACTTCCTCAACAATTCCTTTTCAGTCCGACATCGTACTTCATCAGCAAGTATCTCGCGAATTCTGGCCTGCACAATTTGGAGATCGAACTCTCCAGGAAATTATCAATTCAAAATCATACGTCACATTCCGAGGTAGATTTTTTTTGAACCTATCATTTTTTAAACTCTACATATTGAATTTCAGCTTTGGAATCCACAATATACAAATGAAAATGAGCAAACTACTGCTGGCTGGCATCATTCGTCTCTGTGATTCGGTTTTGTCTCGGAGCAAGAGTAAATTCGATAAAATGGTTGAGAAACAGTAAGTGATTAAAAAAGTTAATCCCGTCAATGAACTTTTTAAAGATTTTTACCGTAATCGTTGCGACTTATATAATTCATGTGGAGTTTTGATGGCAGGATTGAAAAGTGAGTTAAAGAAAATATTCTAAATAGTTTGAGACTTCACTGACTAAGTTTTCATAAATCTTACACAATCCAATTTAAGTTATTAAAAGATTCTAGTGAAAACTTTTTCAGAATCCGTCTACATCTATTGGTCCATGACTGGTCTCCGCACCGAATCCGCGTGGGCCAACCAGTCTCCATCTCCTCCAAGTTATTCCCAGTTGCCGGCACGGACTCACTGATGATTTAACTTTCTCGCAACGTCTTTACAACGTGGCATCCTATTTCAAACAACTGTATATTCATCAACATATTGTGCAACCAAGAATTGATGCAGTTTTCAGGTGAGACCTTTTTTATACTTCCAAACTCTTAAGACAAAGTGAAATTCAGAAATACTACCCAGGAGTCGAGTCTACTTTCGAAATTGAGCGCAACGCATCTATAAACTTCGTGAATACCCCTCCATTTTTGACTTCTCAAGACATACATGCCACGTGTAAATTTCATCGGAGCCATCCAATGCAGAAAACCAAAAGAACTTCCGAAGGTTTCAAATTTTAAAAACTTATTTAAAAAATTATAATATCTGCTAAATTCCAGGAGTTTACCCTTGGATCTCAGCCTATCCTGACGGTTTCGTTGTGCTCTCCACTGGGTTCACTGTTCAATGGAACAAATCTCCGGAGCACATTCGTCAAGCCTATCTGGATACCTTCAGTATCTTCCAAATCTCTTGTCATTTGGCAAACTGGTCTACCAAATTCTTCAAACTTGCCATCAAATCTACTCGTCAAGCCATGGCTTCCACTTCAAGACCTACTAGGTCATCAAAAGTGCCGATGCACGTGTCAC
ATGGAGGATTGAACAGTGTCATTGAATCAGTTTATCACGGAGTTCCAGTTGTTGGAGTACCATTGACTTCTCGTGGTTATGATAATTTACTGAGAATCACTGCACGAGATAGTGGAGTCATGGTGGAAATCGGAGTTCAGTGGGGAAATATTGACTGCCGCTATTAATGAAGTGATTGAAAATGAGAAGTGAGTACATTTAGTTTGATTTCAAAAACGGAAAATTCAGAACGTTCAAATTCAAATAATTGTTTCGATACAAGAAGGAAATGTTAATCTTCCAAGATATGGTCATCGATGTTCCATACACTGAACTTTATCACGCTGCGTTTTGGGTTGAATTCATCGAACGGCACCAGGAAGTCCACACGCTAGAAGTGAGAGATCATTTGAATTTTCTTCAATATTTCCTCGTTGATGTCATTGCCTTCTTCTTCTTTGTCATTTTCTGTACTCTTTCAATTATTTATTACACAATTCGGACAGTTTTCCGAACGATTCGTTCATGGGAACGGAGTTCGGGGAGTTCCTAAAATTGTTTCAAGGGGAAAGAAAAACAATTGATTCATGCATTCATTCATTTCGTTTTAATTAGGATTTCTAGGCATTTCTATGGCGACTACGGGTTTTTTCCAAATTTGATAAACTCTGTGATAAATACATAATTGCTGATGATCAATTTTTTTGAAACTACACATTTAAAAGATCTGCAGAAATCAGTCATATTGTGGCCACATTATGGAGAAATTGATGCGGGTGTATCACAAAGAGAATATACTTTTTCAGTTTTGTCGGTGACTTCTTGAACATAATTTATAGTATACGTTTTCATGGTTCTGTTTGCGGTTGGCGCAAAGAATTCACAAACTTGGTGCATATTGCATTTCCGGTCACATCAGATCAGAAACTTGGTACCTTCAAATTTTTTTATACAAATAAAATTTTCATCAAGTGCATCGTGAATACGAGCTGGCAGGGTTTGGCAAGTTTCTTTAGAATAAACTGACAATCACAGAGTAATTTCTGCTAATTGTTTGAAACATTTTGACTGATTGTGAGTTCATAAAGTTCAAAACTCTTCGTAATATTTGAATGTGTTCTTTCTTATTTTTTCCATCAGAGATATTTCTAGAAAAATACATACACTAGCGTAATATTGAAAGTATACTTTTTGATTTAATCAGTCGGCGACTTCTGTAATTTTGTCTAAAGTATACGTTTTCGGTGTTCTTTATCCGGCTGGGCCAAAGACCTTGCAAACTGGTACCCGGCCATCTTCCAATTTATGGCAGCTCACAAACTTGGCACCTCCCAATGATGAGAGGCTCCACCCACTTACAACCAACTCGTCCGCCCCCCGTTGAGCTCACATGCTTACTCTTTCATTATTCTTCATTTATTTTTGTTACTTGTGTCTATTTTACTGCTGTTTCCGTTTTCTTCTTTCATTATTTATTTAAAATTTTCCATCGCTTACTTCTTTTTCAGTTTAACATGAACTCAGTTTTGAGTAAATCGAAAAACTGATACATACATTCTCCGACGAAATGAATATGTAGCTGAGTCTAAAGACGAGCCGAACAAACTAAATACCAGCCGAAAACTGGAAGTCACCACGAAGAAGAATCAATCAAACAATAAAAAAGACCGCCCCTATAAATAAAGCTCGAAAAAGTCTACCAAGTATTTTTAGAAGAGCTGAGGAAAATAAAGATAGTGCCCAAATTATCACACCAACTGGTAAGTATTTTATCTTTTTGTAATATTTAGTGTTTGTTTCTGACAATTTTAGCATGTTACTAAATTCTTTTCCTTTTCAGGAAGTAAAATTATTGGGAGCCCCTTATATAAACGATCAAAATCGGATTCATTTTTCGACCACGCATTCAATTTGATAAAAATCTTGGAAAGGCTCATTTGGAGAAGTCGTTGCAGCAACATGTCGTTCAACTTCAAAGAAGTTTGCAATCAAAAAAATTCCATTTTCAAAACTTTCAAAAGATCAATATCGCGAAGCAATGGACACATGAATATCCATGTCACCCAAATATTGTGAGATTCCACCAAGCATGGATAGACAAACAAATTCTTCATATTCAATTGGAAATGTGCGATAAAAGTTTGGCGGCATATTGCCATGGAATGATTGGCTGGAAGACAGGAGCTTTGGAATGTGTTCTTGGATATTTTACAAGGATTGGGACATTTACATAATAACTTCATGCTTCACAACGATATCAAACCAGACAATATATTTATGACAAAGAACAAGTTTGTAAACTCGGAGTTTCGGATTAATTTCGGACATGAGATCGGTATGTTTTGTAAATAATTTAAAAATGCACATAAATATTACAGGAACCAATCAATAATTCTTCAAATAAACATTATCAATCGAAGGCGATGGAAAGTACGTTCCAAAGAAGCAATCAATGGTACTCTCTCAATCTTCAGTGATATATTTTCATTTGGAATATCGATTCTGGAAGTAGGAACCAACATTCATCTACCGAGTTATGGGAAGGATGGGAACCAATTAGAAATGGGAAATTCCAGAAGAAATATTGGAACCAATGTCAGATGAACTTCGAGAATTGGTGAAACAAATGATGGACAAAGCCCCGACACGAAGACCAACTTGCTCTGAATAATGAAGAATCATGTAGTGAAGAGAAGCTAAGAATAAGGAAACAAGAAGTTGAACATATTCCTTCGGTTAGTATATTTCAAAATTTAATCCCATAAAATATTACATCTTACTTTACAGGAGTCGCATTTCAACCGATATCCTATCTACAACAGAAAGTCCATTAGGATCACGGGAATCATCTTGCTCATCTATCGATTTTCTTGACCATTTTGAAAATGCTGAAAAAAAGACACCATTTGTGAGTAAGCTGATTTCGATCTAGAAGATGATGAGACGAGTATGAAGTCTTCTCTCCACCAAGAACTCCTGTCAAAAAATCGCGATACCAACAAACAATGCCGGAAGTCAGTCCACCCAGAAAGATTCAAAAGTTATTCAACGGAAAAATTTATTTGATGATAGGAAGGGAATGTAAGTTAACAGGTAAGTTTGCTCTTTTTTCTTTTATAAAATTCGTTTGTTATTTTGACTTTTGAAAATTAAAATTAGGAAATTAAAAAAAGTTATTTAGAAATTAACATATCTCAAAGTGAAAATAGTAATTAGGTGTTTGCAACATCTTAAAAAGCACATTTCACAATTAATAAACTTTCAATTAAAATTGCAATAAAATTACGGGGGAAATTTTAAAATGTCAATTTAAAATAATAATTAATTTTCAGGTCCGTTGTTTTTCATTCAGGTTCAATCGGTTAAATATCAATTCCAGTTTTTAACATCTCATATCATCGATTGGTACAAGTTCTTCCAATCAAAAATCGAGTTCAGTTTTAATCATCACTTTGTTCTTCTTTTTTCTATAAAATAAATTATTTATCACTGAAATTGGTATACAATTTTTATTATACACATTTTAGTTTTAATATTTCAAAAAATCTTTTAAAAGAAAAACAAGGAACCCAGTATAAAATCAGAAAACTGTACGAAATACTGAAAAGTGACGGAGGATTTGGCATATTTTAGGTAAAATTGATAAAAGTTTTGAAGATGTTAAAGCTGATTCAAGTTTTGGGACAGGGGTGGGCAGAAGCTCATATTCTCAAGGGGCGTGGCTTATTTCGTTACCGGAATTCGATTGGTTTTGCTCACAGTTGCTCATGAAAGGGGATGGAACACA 
ATGGGGGGGGGGGGGGGGTGACTAGTAGCACACAAAAAGTGACTGGAATTTCAGAGGGGGAAAATCACAATTTCTTTGGTTGGGGAAGAAGAAGAAAATGAAAAGAGAATGGGAGACAAGTGACTGGTGATTGAAATTAGAATATTTTTGCAAAAAAGTTTAAGCTCGATATGGAGTTGATGACTTGAAAACTACTTGAAGCGTCTTTCCAGCCAAATTTGTGCCATTCAGAGAAAGCATTGCATTATAGGCTCGTGTAGTTGCTCATCGATACAAATGCATATCCTTTGCACTGTTGTGTCAGGTCTCGTAGAATCTCAAATATTTCAAGTAAAAAATTTTCCATTTAAAATTTCTTGCTTACCTTAACATTAACAATGCCCAAACTGTGAGAATAACTGCCATAACAGTGTATCATCAGTATCTGATGAAAGATTATATACGAAAAGGCAATATCCAGCAGTATCTGATGGCGGTAACGTGGCAGCAACTGAAGCTGACAATGAGAAACTGAGCATGAGCTGCTAATCCAGCAACTTGTCCATTTGTTGCCGGAGGGCTCGCTGTTTGTGCAGCATGTTGTTGAGCAACTGCGTGTTGTTGTTGCTGATGCGCTATTAATGAAGCTGTGAATCGGTACGGCTGTTGCTGTGGCAAAAGGGTTCAGGCCAGCAAGAGCATTCAACTGAGACATCTGAAGAAGCGCAGAAGTTGTCAAATAGTCAGCAGGAAGTGTAGCGGTTGGCTGGGATACTGCGTGATGCGGCCATTGGGGAATATCTGAAATATTGGAAATTGGTCTTGAAAATGTAAAAAGAAGAATATACAAACTTGAAAATACAATTATTATCAATCAAGATTTCTTCGGGCTTACCTGTACTTTAAGTAATGGAGCATGGTGCATCGGTCCGATTCCTCCAGCAGTTGCTCTCAGCGTCGGTGCTCCAAGAATTGTGCTCAATGGTACCAGTGTTGTGGCGGCTTGCTGTACGGCTTCCAGATCACTCAGAGTCCTTCGGATTGTTACTTGCTGGATTATTTGCGAATTTCACTGTGATTTGCTCTGAACATCCTGATGGAATACTTCCATTTAGTGTTTTAATTGCAACATCAGCTTCGTCTTTTTTGTCGAAACTACAAATCAACGCCTTTTGAGAGTCCTGAAATAGCTCTAATAAAAAATGAAACAAATTATATTCCGTAAACAATCAACTATATTTTTCTTAATTTTGAAAATTTACACATTTCACATAATTTTAAAACACACCAGTACATTATCTGACAATATTCGAGATGTGATAATCTGTCCAAATGGCCGAAAAATCGATTCCAATTCGTGCAGAGTCATCGATTTTGGAATTCCAGAAACATATAAGTTTGAACCTTTATTTGATCATTGATGGACGGGCATATGATACCTGAAAATTTCAAGTTATAGATTACAATTTTAATATTCTAAAATTTAACTTTGTATTTTTACCAGTGAATGTTTTTCTTTTACATATATGAAGAAGCGAATTTTCAGTAAAAATATAATTAAACATTTTCTAAGCGTTTTGTGCACATCTATTGCTCCTATTAGTGATTGTTTGAAAGACGTGAGACGTTTAACGGACGTTTAGACACACATTTAGAAGCTAAATCATTATAATCGGGAAAGGGGTTTCGCGAAATTAAAGTTGAAATTAAATTTTGAAATTAAAATATTAAAATAAAAAGATTGAAAACTTAAAACAAAAATTGCACAACAGGAGCAAGGGGGTGTTTCTAATAGTCAAGGAACACCCAAACAATTGAAAAATTAAAAACTTTTTTTGAAAATAAAAAAGAGTGAAAGTAAAGTAAAAGACAAGTAGCATGTAAATCAACTAAAAATAATAATATTTCCCCGAAAAAAAAAAAAAAGGTGGAAACCTTGATCGTCTTATCCTGAAATCGCAGGCCATTGATTGAGTTGACTGAACGAAGAGCATCCTCTCCTCGGACGTAATAAACAAACCCGCAACCAGGGGACTGTCTGAAATGAAAAAACCCACTTCCCN2- Clicar na primeira proteína que aparecer.
3- Clicar no NM 063658.6 – Código inicial de acesso ao gene ----Nucleotide sequences
4- Send > File > Formato Fasta (sequences.fasta) --- colocar numa pasta pessoal
5- Abrir com o WordPad
Arquivo Fasta correspondente a versão cDNA, do nosso gene.
6- Clicar no arquivo que contém o contig e arrastar para o Jalview.
7 – Clicar em Colour --- nucleotide
Para mudar o modo de edição clicar em f2 ---- cursor de vermelho fica preto
Usa-se o Jalview para:
Versão genômica com 8 000 pb, para retirar a seq de interesse 1115 a 3397 (3394 + UGA – códon de parada) 
8- Incluir o código de terminação UGA, shift e clica lá em cima (selecionar)
9- Após selecionar a sequência, Edit > copy ou control c + control (colar wordpad)
Nesse ponto temos os éxons e os íntrons
Est2genome
Versão nucleotídeo pós-splicing – cDNA
Caixa de texto colocar versão Cdna – Spliced EST
Na caixa de baixa colocar o contig (genoma) em baixa
Selciona Show de Alignment Yes
Pode mandar o resultado para o email
Run est2genome
Gera Output file
Seq minúsculas são íntrons
Importante para desenhar primers para amplificar apenas RNA (junção éxon – éxon)
Importante para esquematizar genes
CDS – CODEN DNA SEQUENCES