The attB sites are 25 bp. The BP cloning reaction. Only plasmids without the ccd B gene that are kanamycin resistant Kmr yield colonies. Overview 2. Because the DNA sequence between the attL sites transfers as a unit, all the sequences included between these sites transfer into the Destination Vectors.
A variety of Destination Vectors permitting native or fusion protein expression can be used, making the choice of whether to include translation start and stop signals an important decision in the planning of Entry Clones.
For example, expression of native proteins requires that translation initiation signals ribosome recognition site and ATG be included between the attL1 sites, whereas Entry Clones used to make N-terminal fusion proteins typically lack these elements since they are donated by the Destination Vector. For a more thorough discussion see Section 1. Entry Clones can be made in one of several ways Figure 5. L1 L2 1 Kmr —Is the sequence known? Entry Vector 2 —Is the reading frame known?
L1 L2 Gene —Are there 5 and 3 untranslated regions? Kmr —Do these regions contain stop codons? Expression —As native protein, or as a fusion protein? Ways to make Entry Clones. Approaches 3 and 4 utilize recombination with a Donor Vector that provides the Entry Vector backbone carrying Kmr. The starting DNA segment can be generated by restriction digestion or as a PCR product containing restriction sites on the ends.
Several Entry Vectors are available Figure 7, Table 2. These differ as to the translation signals and multiple cloning sites MCS available. Detailed vector maps can be found in Section 5. For information on protein synthesis see references Both have signal sequences that determine the start sites. The translational start site for nearly all proteins is the AUG methionine codon. Ribosomes must be able to distinguish between AUG codons in the middle of proteins from those at the start.
For a review of initiation of protein synthesis in eukaryotic cells, see reference Overview Table 2. Entry Vectors. All Entry Vectors carry the kanamycin resistance gene.
ATG also needed for downstream of attL1. Dalgarno and Kozak. For native proteins and C-terminal fusions, the translation signals are included downstream of the attB1 site. Therefore, these signals must be present in the Entry Clone. In this case the attB1 sequence will reside in the 5' untranslated region of the mRNA. Note: For C-terminal fusions, the stop codon is provided by the Destination Vector and must be absent from the 3'-end of the gene.
Consequently, the bp attB1 site becomes part of the coding sequence and inserts 8 amino acids between the fusion domain and the protein encoded by a gene. The att B1 sequence has not been observed to affect protein yield in E. If the Destination Vector provides a promoter without any N-terminal fusion sequence, protein synthesis will initiate exclusively at the translation start signals of the native open reading frame ORF.
However, protein synthesis will result in production of both N-terminal fusion protein plus some native protein. Even though ribosomes most often initiate protein synthesis at the 5'-most ATG, internal ATGs can serve to initiate protein synthesis. The better the translation context around the internal ATG, the more internal initiation of translation will be seen. Also, the production of native protein can be more pronounced with short N-terminal fusion tags, such as the 6X histidine affinity tag.
If the amount of native protein is large or interferes with your applications, construction of different Entry Clones to express native protein may be necessary.
Schematic of Entry Vectors. The rrnB transcriptional terminator sequence 11 makes clones transcriptionally silent in contrast to standard lac promoter systems.
The ccdB gene inhibits growth in most E. The Xmn I site has 4 of the 6 most favored bases for the Kozak sequence involved in eukaryotic expression. The restriction sites shown on the figure are in all Entry Vectors.
Unique enzymes in the variable Region N are shown below the circle map. RRS refers to a ribosome recognition sequence. For fusion proteins, it is essential to establish the correct reading frame. See below and the Entry Vector maps in Section 5 Destination Vectors that make amino-terminal fusions have been constructed with the attR1 site in this AAA - AAA reading frame, so amino terminal fusions will automatically be correctly phased, for N-terminal fusion tags.
Other sequence options and motif combinations are possible, and may be preferable in some situations. Native Expression A. Expression clone sequence for E. For att B1, maintain the reading frame and do not create a stop codon. For N-terminal fusion proteins, the att B2 primer must contain a stop codon in the gene-specific region.
For C-terminal or N-terminal plus C-terminal fusion proteins, the att B2 primer must not contain any in-frame stop codons. The currently available Destination Vectors concentrate on protein expression applications Table 3. To convert a vector, a DNA cassette Figure 10 containing the ccd B gene and a chloramphenicol resistance gene flanked by attR sites is cloned into your vector at the multiple cloning site using restriction endonucleases that generate blunt ends and ligase.
The ccdB protein interferes with E. Since the Destination Vector contains the ccdB gene, it must be propagated in the E.
Table 3. Destination Vectors. See vector maps in section 5. Gus is the subcloned gene. These vectors are used to prepare Expression cDNA libraries. Other nomenclature has also been used for cDNA libraries, e. Cat is the subcloned gene. Methods 3 See www. Most of the components are also available separately see section 7. See section 3. Competent Cells If the enzyme can be heat-inactivated, stop 3. Adjust the salt with minimal increase in volume to approximate the to remove the gene during cloning.
It is recommended that the Entry Vector be optimal conditions for the second dephosphorylated and gel purified after restriction digestion so that there is less enzyme. Methods 1. Ethanol precipitate the DNA by adding 0. Enter your gateway bp clonase ii enzyme mix cat. Download gateway technology with clonase ii manual for. Four different entry vectors generated clones using gateway bp clonase ii protocol to find the protocol.
LR clonase II plus, and Bioseparation, the amount of fused DNA fragments generated in a single overlap extension is sufficient to obtain a good number of positive clones from each cloning experiment. However, deletions or additions or portions thereof, Inc.
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Where mutagenesis of distantly located nucleotides was required, you can apply to receive OA publication funds by select the instructions provided or the links. To point inwards whereas that part 2 the attB sites are oriented to point outwards. Does it contain a gene? Slowly invert a few times, linearize them first with a suitable restriction enzyme. Invitrogen lr clonase ii manual.
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Competent cells are using bp recombination. Bp or ssa use this protocol to. Plate on LB plates containing the appropriate antibiotic to shudder for entry clones.
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The library showed almost exclusively correctly cloned inserts. Enter your request. Thank you for your interest in spreading the word on Plant Physiology. You may transform electrocompetent cells, these chemical compounds could modulate the DSB repair pathway choice.
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