When the bacteriophage lambda enters and exits the lysogenic cycle, it utilizes a variety of recombinases to insert the viral genome into the host DNA. The Gateway system uses modified versions of these proteins that allow for highly site specific recombination at very high efficiencies. This is a summary of the information that can be found about gateway cloning on the Invitrogen web site.
The recognition sites for these enzymes are called att sites. When the phage DNA is being inserted during the BP reaction, attB and attP sites are recombined. This reaction will swap the DNA between strands, creating an attL and an attR site. When phage DNA is excised during the LR reaction, attL and
attR sites are similarly recombined, yielding attB and attP sites (Figure 1). There are four specific sets of
att sites, notated 1-4. These sites will only recombine within the group: for example, attL1 sites will only recombine with attR1 sites, yielding attB1 and attB2 sites. These sites are also orientation specific.
Creating a gateway strategy requires knowledge of the att sites required.
Figure 1. The BP and LR reaction cycle.
In order to be able to select for the correct clone, the Gateway system uses a combination of drug selectivity and the killer gene, ccdB. ccdB encodes a bacteriotoxin that prevents DNA replication. All of the backbones for Gateway clones carry this gene, and only bacteria carrying ccdA, the antidote gene,

will grow when transformed with these plasmids. NOTE: Only ccdA competent cells can be used to grow up these clones!
In order to easily talk about the steps in Gateway cloning, names have been assigned to different types of vectors.
Donor Vector (ccdB +, KanR): The donor vector provides the backbone for the creation of entry clones.
Donor vector are typically denoted with names starting in pDONR. These plasmids carry B or P sites flanking the ccdB gene, as well as the kanamycin resistance gene.
Entry Vector (ccdB -, KanR): The entry vectors are created when the donor vector undergoes the BP reaction with a PCR product or expression clone containing a sequence of interest. Entry vectors contain L or R sites flanking a seqeuence of interest, as well as the kantamycin resistance gene. The power of gateway cloning lies in the ability to build large libraries of entry vectors, allowing for rapid mixing and matching of promoters and open reading frames.
Destination Vector (ccdB+, AmpR): The destination vector provides the backbone for expression clones.
Destination vectors are typically denoted with names starting in pDEST. These plasmids carry L or R sites flanking the ccdB gene, as well as the ampicillin resistance gene.
Expression Clone (ccdB-, AmpR): The expression clone is the final product of a gateway reaction. It is the complete plasmid, ready for injection.

Figure 2. Strategy for crating entry vectors.
The entry vector is the basic building block of a gateway clone. Entry vectors will be recombined into a complete expression vector into three slots: slot I, II or III. attB sites can be added to your DNA of interest using primers containing the attB sites and PCR amplification. The sites added and the donor vector used depend on which slot the entry vector is for.
Slot
I
II
III
Typical uses
Promoter
ORF, flip cassette
3’ UTR, ORF
att sites attP4 – attP1R attB1-attB2 attP2-attP3 pDONR pDONR P4 P1R pDONR 221 pDONR p2R p3
The BP reaction will swap the DNA on interest with the ccdB gene. On kanamycin plates, the only viable tranformants of the BP reaction mix will be those carrying the kanamycin resistance gene and no ccdB gene, and thus the only viable colonies will be carrying the entry vector.

A guide for how to design primers primers can be seen on page 12. Once you amplify your product, add
the following together and vortex.
PCR Reaction 25 μL dH2O 65 μL
10X TE (In protocol book) 10 μL
30% PEG, 30 mM MgCl2 (Provided with kit) 50 μL
Centrifuge for 15 minutes at full speed. Carefully remove supernatant and resuspend the DNA in TE 10
μL 1X TE, or to greater than 150 ng/μL.
For the gateway reaction, combine the following on ice: dH20 To 10 μL
10 X TE 1 μL
Donor vector 150 ng
PCR insert 250 ng
BP clonase II 2 μL
Incubate at room temperature overnight. The next day, add 1 μL proteinase K and incubate for 10 minutes at 37° C. Long transform the entire reaction using the standard protocol and plate 50 and 100
μL of cells onto kanamycin plates. For a positive control, use 150 ng of pDONR 221 and 2 μL pEXP7-tet.

Figure 3. Using the LR reaction to create entry clones.
Combining multiple entry vectors with a destination vector allows us to create expression clones carrying multiple specific elements. Only the complete recombinant will both loose the ccdB gene and gain ampicillin resistance.
To do the LR reaction, combine the following on ice. Vortex and spin down the contents at incubate at room temperature overnight. dH20 To 10 μL
10 X TE 1 μL
Destination vector 150 ng

Slot I entry vector 150 ng
Slot II entry vector 150 ng
Slot III entry vector 150 ng
LR clonase II 2 μL
The next day, add 1 μL proteinase K and incubate for 10 minutes at 37° C. Long transform the entire reaction using the standard protocol. The LR reaction has a much lower efficiency than the BP reaction, so following the long reaction spin down the bacteria at >30,000 RPM for 3 minutes and resuspend in
200 μL LB. Plate 50 and 100 μL of cells onto ampicillin plates. For a positive control, use 150 ng of pDEST
221 AD and 2 μL pENTR-gus.

For each set, the sequences of the recombination sites and the places where they combine are essential for creating constructs. The sequences can be accessed using the polylinker files available on
Invitrogen’s web site, however they are confusing, hard to find, and sometimes hard to follow. Here is a color marked copy of these sequences.

attB1
---ACAGTTT GTACAAAAAAGCAGGCT---
---TGTCAAACATGTTT TTTCGTCCGA--- attP1
---CAAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACACATTGATGAGCAA
---GTTTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTGTAACTACTCGTT
TGCTTTTTTATAATGCCAACTTTGTACAAAAAAGTTGAACGAGAAACGTAAAATGATATAAATA
ACGAAAAAATATTACGGTTGAAACATGTTTTTTCAACTTGCTCTTTGCATTTTACTATATTTAT
TCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATAT
AGTTATATAATTTAATCTAAAACGTATTTTTTGTCTGATGTATTATGACATTTTGTGTTGTATA
GCAGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTAG---
CGTCAGTGATACTTAGTTGATGAATCTACCATAATCACTGGACATC--- attL1
---CAAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACACATTGATGAGCAA
---GTTTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTGTAACTACTCGTT
TGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCT---
ACGAAAAAATATTACGGTTGAAACATGTTTTTTCGTCCGA--- attR1
---ACAGTTT GTACAAAAAAGTTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAA
---TGTCAAACATGTTT TTTCAACTTGCTCTTTGCATTTTACTATATTTATAGTTATATAATTT
TTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATGCAGTCACTATGA
AATCTAAAACGTATTTTTTGTCTGATGTATTATGACATTTTGTGTTGTATACGTCAGTGATACT
ATCAACTACTTAGATGGTATTAGTGACCTGTAG---
TAGTTGATGAATCTACCATAATCACTGGACATC---

attB2
---ACCCAGCTTTCTTGTACAAAGTGGT---
---TGGGTCGAAAGAACATGTTTCACCA--- attP2
---CATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCT
---GTATCACTGACCTATACAACACAAAATGTCATAATACATCAGACAAAAAATACGTTTTAGA
AATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTT CTTGTACAAAGTTGG
TTAAATTATATAACTATAAATATAGTAAAATGCAAAGAGCAAGTCGAAAGAACATG TTTCAACC
CATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAA
GTAATATTCTTTCGTAACGAATAGTTAAACAACGTTGCTTGTCCAGTGATAGTCAGTTTTATTT
ATCATTATTTG---
TAGTAATAAAC--- attL2
---ACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAA
---TGGGTCGAAAGAACATGTTTCAACCGTAATATTCTTTCGTAACGAATAGTTAAACAACGTT
CGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTG---
GCTTGTCCAGTGATAGTCAGTTTTATTTTAGTAATAAAC--- attR2
---CATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCT
---GTATCACTGACCTATACAACACAAAATGTCATAATACATCAGACAAAAAATACGTTTTAGA
AATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTT CTTGTACAAAGTGGT
TTAAATTATATAACTATAAATATAGTAAAATGCAAAGAGCAAGTCGAAAGAACATG TTTCACCA
---
---

attB3
---CAACTTTATTATACATAGTTG---
---GTTGAAATAATATGTATCAAC--- attP3
---CCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATC
---GGTATCACTGACCTATACAACACAAAATGTCATAATACATCAGACAAAAAATACGTTTTAG
TAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAACTTT ATTATACAAAGTTG
ATTAAATTATATAACTATAAATATAGTAAAATGCAAAGAGCAAGTTGAAATAATATG TTTCAAC
GCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAA
CGTAATATTTTTTCGTAACGAATAGTTAAACAACGTTGCTTGTCCAGTGATAGTCAGTTTTATT
AATCATTATTT---
TTAGTAATAAA--- attL3
---CAACTTTATTATACAAAGTTGGCATTATAAAAAAGCATTGCTTATCAATTTGTTGCAACGA
---GTTGAAATAATATGTTTCAACCGTAATATTTTTTCGTAACGAATAGTTAAACAACGTTGCT
ACAGGTCACTATCAGTCAAAATAAAATCATTATTT---
TGTCCAGTGATAGTCAGTTTTATTTTAGTAATAAA--- attR3
---CCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATC
---GGTATCACTGACCTATACAACACAAAATGTCATAATACATCAGACAAAAAATACGTTTTAG
TAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAACTTT ATTATACATAGTTG
ATTAAATTATATAACTATAAATATAGTAAAATGCAAAGAGCAAGTTGAAATAATATG TATCAAC
---
---

attB4
---CAACTTTGTATAGAAAAGTTG---
---GTTGAAACATATCTTTTCAAC--- attP4
---AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATG
---TTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTTTAACTATTCGTTAC
CTTTCTTATAATGCCAACTTT GTATAGAAAAGTTGAACGAGAAACGTAAAATGATATAAATATC
GAAAGAATATTACGGTTGAAACATATCT TTTCAACTTGCTCTTTGCATTTTACTATATTTATAG
AATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCC
TTATATAATTTAATCTAAAACGTATTTTTTGTCTGATGTATTATGACATTTTGTGTTGTATAGG
AGTCACTATGAATCAACTACTTAGATGGTATTAGTGACCTGTA---
TCAGTGATACTTAGTTGATGAATCTACCATAATCACTGGACAT--- attL4
---CAACTTTGTATAGAAAAGTTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAA
---GTTGAAACATATCTTTTCAACTTGCTCTTTGCATTTTACTATATTTATAGTTATATAATTT
TTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGA
AATCTAAAACGTATTTTTTGTCTGATGTATTATGACATTTTGTGTTGTATAGGTCAGTGATACT
ATCAACTACTTAGATGGTATTAGTGACCTGTA---
TAGTTGATGAATCTACCATAATCACTGGACAT--- attR4
---AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATG
---TTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTTTAACTATTCGTTAC
CTTTCTTATAATGCCAACTTT GTATAGAAAAGTTG---
GAAAGAATATTACGGTTGAAACATATCT TTTCAAC---

The proper design of attB-attached primer is a crucial point for the BP and LR reaction of Gateway clone and the functional expression of the gene. The following is a simplified way to make your gateway primers.
According to your gateway entry vector(s), choose appropriate attB adapter pair(s).
1) attB1: 5’GGGGACAAGTTTGTACAAAAAAGCAGGCT TC XXXXXX(18-25nt)-3’ (Added to 5’ PCR forward primer)
2) attB1: 5’GGGGCTGCTTTTTTGTACAAACTTG T XXXXXX(18-25nt)-3’ (Added to 3’ PCR reverse primer)
3) attB2: 5’GGGGCAGCTTTCTTGTACAAAGTGG GA XXXXXX(18-25nt)-3’ (Added to 5’
PCR forward primer)
4) attB2: 5’GGGGACCACTTTGTACAAGAAAGCTGGGT T XXXXXX(18-25nt)-3’ (Added to
3’ PCR reverse primer)
5) attB3: 5’GGGGCAACTTTGTATAATAAAGTTG T XXXXXX(18-25nt)-3’ (18-25nt)Added to 3’ PCR reverse primer)
6) attB4: 5’GGGGCAACTTTGTATAGAAAAGTTG GA XXXXXX(18-25nt)-3’ (Added to 5’
PCR forward primer)
Red : is the sequences of attB sites that function as adapters.
Green : is the the nucleotide(s) added to keep the sequence in the right reading frame. You can change it to other nucleotide(s) if you don’t like it (but do not choose stop code).
XXXXXX(18-25nt): Your specific gene sequence.
Highlighted Yellow : If you have universal attB adapters available in your fridge, you can only choose highlighted yellow sequence to do 1 st
PCR then add the corresponding universal attB primers to do the 2 nd
PCR. Taken from http://www.med.upenn.edu/lamitinalab/documents/Gatewaycloneprimerdesign.pdf