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