Gateway Cloning Tutorial: Overview: Simulating Gateway multisite cloning

FisherScientific provide a number of specialised pDONR vectors that allow multiple "parts" (2, 3 or 4) to be recombined, in a specific order and orientation, with a destination vector. The Geneious Prime Gateway Cloning tool allows simulation of multisite Gateway reactions.

See the MultiSite Gateway® Pro manual for detailed information on multisite Gateway cloning.

Depiction of steps required in 3 part Gateway multisite cloning. Picture taken from MultiSite Gateway® Pro manual


The steps required for multisite cloning parallel those performed in Exercises 1, 2 and 3:

  1. Design primers for PCR amplification and addition of unique attB sites to each part.
  2. Create individual entry clones for each part using specific multisite pDONR vectors.
  3. Recombine the 3 entry clones with a destination vector using the Gateway cloning tool.

The Gateway clonase recognition sites are directional and must be orientated appropriately in order for multisite recombination to proceed correctly. If you examine the above figure you will see that in a 3 part clonase reaction the PCR products for parts 1 & 3 have attB sites oriented to point inwards, whereas for part 2 the attB sites are oriented to point outwards.

For multisite gateway cloning, variants of the attB recognition site are used for each part, in the case of a 3 part reaction: attB1+attB4, attB4R+attB3R and attB3+attB2.

The following table is provided to assist when using the Geneious primer design tool to ensure you select the correct att sites when adding to primers for amplification of multisite Gateway parts.


Vector and recombination type left/right att sites att site to add to forward primer Att site to add to reverse primer
pDONR221 P1-P5R
2-fragment - part 1 attB1 Sense: attB1
4-fragment - part 1 attB5r Antisense: attB5r
pDONR2221 P5-P2
2-fragment - part 2 attB5 Sense: attB5
attB2 Antisense: attB2
pDONR221 P1-P4
3-fragment - part 1 attB1 Sense: attB1
attb4 Antisense: attB4
pDONR221P4R-P3R
3-fragment - part 2 attB4r Sense: attB4r
4-fragment - part 2 attB3r Antisense: attB3r
pDONR221 P3-P2
3-fragment - part 3 attB3 Sense: attB3
4-fragment - part 3 attB2 Antisense: attB2
pDONR221 P5-P4
4-fragment - part 4 attB5 Sense: attB5
attB4 Antisense: attB4

Go to Exercise 4 to perform the steps required to simulate a multisite Gateway reaction.


General Overview: Overview of tutorial
Exercise 1: Designing primers for Gateway cloning
Exercise 2: Simulating an "BP" Entry clone reaction
Exercise 3: Simulating a "LR" Destination clone reaction
Multisite Overview: Overview of Gateway multisite cloning
Exercise 4: Multisite Part 1: Primer design and PCR
Exercise 5: Multisite Part 2: Creation of Entry and Destination vectors
Gateway Vectors: Preparing Gateway vector sequences

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