T7. Benchling
Abajo encontrará un Laboratorio para aprender a usar benchling
-El link a Benching es https://benchling.com
- Si se va a utilizar un ensamblaje por bloques, debe recordar añadir los prefijos y sufijos
Para añadir piezas adelante (en el prefijo EcoRI-XbaI)
- La secuencia a insertar se corta con EcoRI- SpeI
- El plásmido o sitio donde se ingresa se corta en EcoRI-XbaI
• (XbaI y SpeI se unen de manera complementaria)
Para añadir piezas atrás (en el sufijo SpeI-PstI)
- La secuencia a insertar se corta con XbaI- PstI
- El plásmido o sitio donde se ingresa se corta en SpeI- PstI
• (XbaI y SpeI se unen de manera complementaria)
Estos son los prefijos y Sufijos que bordean la secuencia que queremos insertar
Protein Coding Region Prefix*
GAATTC GCGGCCGC T TCTAG[ATG Remaining CDS]
EcoRI NotI XbaI
Protein Coding Region Suffix
[CDS]T ACTAGT A GCGGCCG CTGCAG
SpeI NotI PstI
Este es el prefijo para otras partes que no son CDS
- 5' GAATTCGCGGCCGCTTCTAGAG '3 - for all other parts
Más detalle en http://parts.igem.org/Help:Prefix-Suffix
Un ejemplo es este CDS que está en color ROJO y al cual le añadimos el prefijo GAATTCGCGGCCGCTTCTAG y el sufijo TACTAGTAGCGGCCGCTGCAG
EcoRI
GAATTCGCGGCCGCTTCTAGATGGGACATGTTACATCTCCCTCTAAACGCTATCCTGCCTCTTTTAAGCGGGCAGGTAGTATTCTTGGCGTGAGTATTGCGCTTGCTGCATTTTCCAATGTGGCAGCAGCGGGTTGTGAGTATGTGGT TACCAATAGCTGGGGGTCGGGATTTACCGCAGCAATTCGTATTACTAACTCAACATCTTCTGTAATCAATGGTTGGAACGTCAGCTGGCAATATAACAGCAACCGTGTTACCAACTTATGGAATCCCAACCTTTCCGGTAGCAATCCCTATACGGCGTCCAA CCTGAGTTGGAATGGCACTATTCAACCTGGGCAAACGGTAGAGTTTGGTTTCCAGGGGGTTACCAATAGCGGCACTGTCGAGAGTCCAACAGTGAATGGTGCTGCATGTACTGGCGGAACCAGTTCTTCGGTGAGTTCTTCCAGTGTTGTGAGTTCAAGCTC GTCATCGCGTAGCAGTGTGTCTTCAAGCTCTGTTGTGTCCTCAAGCTCCAGCGTAGTGAGTTCATCGTCTTCCTCTGTGGTCAGCGGCGGCGGCCAGTGTAATTGGTATGGAACCCTGTATCCGTTGTGTGTGAGCACAACCTCTGGTTGGGGTTACGAAAA CAACAGAAGCTGTATCTCTCCATCAACCTGTTCGGCCCAGCCGGCGCCTTATGGGATTGTCGGGGGCTCGAGCTCGCCCAGCTCAATTTCCAGTTCAAGCGTCCGCTCCAGCAGCTCGTCTTCTGTAGTACCGCCTAGCAGTAGCTCATCTTCCAGTGTTCC ATCAAGCAGCTGTTCCAGTGTTAGCTCCAGCTCGGTTGTCTCTTCCAGTTCATCTTCTGTGAGTGTGCCCGGAACCGGTGTGTTCCGTGTTAATACTAAGGGTAACCTGACGAAAGACGGTCAACTGCTGCCTGCGCGTTGCGGGAACTGGTTTGGTTTGGA GGGGCGTCATGAGCCATCAAATGATGCCGATAACCCCAGCGGTGCGCCAATGGAGTTGTATGCGGGTAACATGTGGTGGGTGAATAACAGCCAAGGTTCCGGTCGCACCATTCAGCAGACTATGACTGAGTTGAAGCAGCAGGGTATTACTATGTTGCGTCT GCCAATTGCACCGCAAACCCTGGATGCAAATGACCCGCAAGGTCGCAGTCCAAACCTCAAAAACCATCAATCCATTCGTCAATCCAACGCGCGTCAAGCATTGGAGGATTTCATCAAACTGGCTGATCAAAATGACATCCAGATCTTTATTGATATCCACTC CTGCTCTAATTACGTTGGTTGGCGGGCCGGTCGTTTGGATGCCCGTCCGCCCTATGTGGATGCGAATCGCGTTGGTTACGACTTCACTCGTGAAGAGTATTCCTGTTCTGCTACCAATAACCCCAGTTCTGTTACCAGGTTCCATGCTTACGATAAGCAGAAG TGGTTGGCAAACCTGCGTGAAATCGCCGGACTATCCGCCAAGCTGGGGGTAAGTAACCTGATTGGTATTGATGTCTTCAATGAGCCTTATGATTACACTTGGGCAGAATGGCAGGGTATGGTTGAAGAGGCCTATCAGGCGATCAATGAAGTTAACCCCAATA TGCTTATTATCGTTGAAGGTATTTCCGCCAATGCTAATACGCAAGATGGAACACCTGACACATCCGTACCTGTGCCACACGGTAGCACCGACTTGAATCCAAACTGGGGTGAAAACCTCTACGAAGCGGGTGCTAACCCACCCAACATTCCCAAGGATCGCCT GTTGTTCTCTCCACACACTTATGGTCCGTCCGTGTTTGTTCAAAGACAATTCATGGAACCGGCGCAGACAGAGTGTGCAGGGCTGGAAGGTGATGAAGCAGCTCAGGCCAGGTGCGGTATTGTGATTAATCCGACCGTGCTTGAGCAAGGTTGGGAAGAGCAC TTTGGCTATCTGCGTGAATTGGGTTACGGTATTTTGATTGGTGAATTTGGCGGTAATATGGATTGGCCTGGTGCCAAGTCGAGCCAGGCTGACCGTAATGCCTGGAGCCATATCACCACCAACGTTGACCAGCAATGGCAACAGGCGGCGGCAAGCTATTTCA AGAGGAAAGGGATAAATGCTTGCTACTGGTCGATGAACCCTGAATCAGCAGATACCATGGGTTGGTATTTAACTCCCTGGGATTCAGTGACTGCCAACGATATGTGGGGTCAGTGGACAGGTTTCGATCCTCGTAAAACCCAGCTGTTGCACAATATGTGGGG TTTGTAATACTAGTAGCGGCCGCTGCAG
SpeI
Si lo tuviésemos que clonar en un plásmido que se corta con EcoRi y XbaI
>pSB1C3 Part-only sequence (2070 bp)
tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctc ggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaa aaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctgg aagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccac tggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacctt cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttc tacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtat atatgagtaaacttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacag gagtccaagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgaga cgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatg aaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgt gaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctt tacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattat ggtgaaagttggaacctcttacgtgcccgatcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaa aaatccttagctttcgctaaggatgatttctggaattcgcggccgcttctagag
EcoRI XbaI
Entonces eliminamos el fragmento interno y los sustituimos por nuestro inserto
>pSB1C3 Part-only sequence (2070 bp)
tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctc ggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaa aaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctgg aagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccac tggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacctt cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttc tacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtat atatgagtaaacttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacag gagtccaagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgaga cgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatg aaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgt gaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctt tacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattat ggtgaaagttggaacctcttacgtgcccgatcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaa aaatccttagctttcgctaaggatgatttctggaattcgcggccgcttctagag
EcoRI XbaI
>pSB1C3 Part-only sequence (2070 bp)
tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctc ggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaa aaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctgg aagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccac tggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacctt cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttc tacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtat atatgagtaaacttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacag gagtccaagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgaga cgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatg aaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgt gaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctt tacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattat ggtgaaagttggaacctcttacgtgcccgatcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaa aaatccttagctttcgctaaggatgatttctggaaTTCGCGGCCGCTTCTAGATGGGACATGTTACATCTCCCTCTAAACGCTATCCTGCCTCTTTTAAGCGGGCAGGTAGTATTCTTGGCGTGAGTATTGCGCTTGCTG CATTTTCCAATGTGGCAGCAGCGGGTTGTGAGTATGTGGTTACCAATAGCTGGGGGTCGGGATTTACCGCAGCAATTCGTATTACTAACTCAACATCTTCTGTAATCAATGGTTGGAACGTCAGCTGGCAATATAACAGCAACCGTGTTACCAACTTATGGA ATCCCAACCTTTCCGGTAGCAATCCCTATACGGCGTCCAACCTGAGTTGGAATGGCACTATTCAACCTGGGCAAACGGTAGAGTTTGGTTTCCAGGGGGTTACCAATAGCGGCACTGTCGAGAGTCCAACAGTGAATGGTGCTGCATGTACTGGCGGAACCA GTTCTTCGGTGAGTTCTTCCAGTGTTGTGAGTTCAAGCTCGTCATCGCGTAGCAGTGTGTCTTCAAGCTCTGTTGTGTCCTCAAGCTCCAGCGTAGTGAGTTCATCGTCTTCCTCTGTGGTCAGCGGCGGCGGCCAGTGTAATTGGTATGGAACCCTGTATC CGTTGTGTGTGAGCACAACCTCTGGTTGGGGTTACGAAAACAACAGAAGCTGTATCTCTCCATCAACCTGTTCGGCCCAGCCGGCGCCTTATGGGATTGTCGGGGGCTCGAGCTCGCCCAGCTCAATTTCCAGTTCAAGCGTCCGCTCCAGCAGCTCGTCTT CTGTAGTACCGCCTAGCAGTAGCTCATCTTCCAGTGTTCCATCAAGCAGCTGTTCCAGTGTTAGCTCCAGCTCGGTTGTCTCTTCCAGTTCATCTTCTGTGAGTGTGCCCGGAACCGGTGTGTTCCGTGTTAATACTAAGGGTAACCTGACGAAAGACGGTC AACTGCTGCCTGCGCGTTGCGGGAACTGGTTTGGTTTGGAGGGGCGTCATGAGCCATCAAATGATGCCGATAACCCCAGCGGTGCGCCAATGGAGTTGTATGCGGGTAACATGTGGTGGGTGAATAACAGCCAAGGTTCCGGTCGCACCATTCAGCAGACTA TGACTGAGTTGAAGCAGCAGGGTATTACTATGTTGCGTCTGCCAATTGCACCGCAAACCCTGGATGCAAATGACCCGCAAGGTCGCAGTCCAAACCTCAAAAACCATCAATCCATTCGTCAATCCAACGCGCGTCAAGCATTGGAGGATTTCATCAAACTGG CTGATCAAAATGACATCCAGATCTTTATTGATATCCACTCCTGCTCTAATTACGTTGGTTGGCGGGCCGGTCGTTTGGATGCCCGTCCGCCCTATGTGGATGCGAATCGCGTTGGTTACGACTTCACTCGTGAAGAGTATTCCTGTTCTGCTACCAATAACC CCAGTTCTGTTACCAGGTTCCATGCTTACGATAAGCAGAAGTGGTTGGCAAACCTGCGTGAAATCGCCGGACTATCCGCCAAGCTGGGGGTAAGTAACCTGATTGGTATTGATGTCTTCAATGAGCCTTATGATTACACTTGGGCAGAATGGCAGGGTATGG TTGAAGAGGCCTATCAGGCGATCAATGAAGTTAACCCCAATATGCTTATTATCGTTGAAGGTATTTCCGCCAATGCTAATACGCAAGATGGAACACCTGACACATCCGTACCTGTGCCACACGGTAGCACCGACTTGAATCCAAACTGGGGTGAAAACCTCT ACGAAGCGGGTGCTAACCCACCCAACATTCCCAAGGATCGCCTGTTGTTCTCTCCACACACTTATGGTCCGTCCGTGTTTGTTCAAAGACAATTCATGGAACCGGCGCAGACAGAGTGTGCAGGGCTGGAAGGTGATGAAGCAGCTCAGGCCAGGTGCGGTA TTGTGATTAATCCGACCGTGCTTGAGCAAGGTTGGGAAGAGCACTTTGGCTATCTGCGTGAATTGGGTTACGGTATTTTGATTGGTGAATTTGGCGGTAATATGGATTGGCCTGGTGCCAAGTCGAGCCAGGCTGACCGTAATGCCTGGAGCCATATCACCA CCAACGTTGACCAGCAATGGCAACAGGCGGCGGCAAGCTATTTCAAGAGGAAAGGGATAAATGCTTGCTACTGGTCGATGAACCCTGAATCAGCAGATACCATGGGTTGGTATTTAACTCCCTGGGATTCAGTGACTGCCAACGATATGTGGGGTCAGTGGA CAGGTTTCGATCCTCGTAAAACCCAGCTGTTGCACAATATGTGGGGTTTGTAATActagag