If desired, a Gateway cloning vector may be constructed manually and then added to the Cloning Vector Catalog prior to beginning the cloning workflow. Prepare the vector as follows and refer to the example at the bottom of this topic.

  • Set the type to circular by selecting Sequence > Circular.
  • Add misc_recomb features to your sequence on the appropriate sub-sequences where recombination occurs. Note that while it is preferred for the entire recombination site to exist within your sequence, SeqBuilder Pro at minimum requires the appropriate att core sequence for each of the five types of recombination sites. SeqBuilder Pro uses these core sequences along with the feature labels to correctly identify each Gateway plasmid:
    • Type 1: GTACAAA (forward strand)

    • Type 2: CTTGTAC (reverse strand)

    • Type 3: ATTATAC (reverse strand)

    • Type 4: GTATAGA (forward strand)

    • Type 5: GTATACA (forward strand)
  • Label the features with the following /dnas_title tags:
    • For Expression Clones: attB1, attB2, attB3, attB4, and attB5

    • For Donor Vectors: attP1, attP2, attP3, attP4, and attP5

    • For Entry Clones: attL1, attL2, attL3, attL4, and attL5

    • For Destination Vectors: attR1, attR2, attR3, attR4, and attR5


The pAd-PL-DEST sequence provided in SeqBuilder Pro’s Vector Catalog is shown below as an example of how to use these tags:

misc_recomb 512..636




      /note=“recombination site for the Gateway® LR reaction”

misc_recomb complement(2092..2216)




      /note=“recombination site for the Gateway® LR reaction”

After creating and saving the vector, add it to the Cloning Vector Catalog so it can be used with future projects.

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