Construction of expression vectors of (fluorescent) proteins with NBRPvectorsYasunori Sasakura 15th December 2017
Modified on 9th March 2021
Modified on 26th January 2023
Most NBRP vectors for expression of a protein, including fluorescent protein, fusion
protein, and Ciona protein (excluding TALENs), share a backbone design (Figure1). The vectors are named “pSPxxxx”. pSP is the vector backbone, and xxxx
corresponds to the arbitrary ORF inserted in the pSP vector.
(Caution: The above names of NBRP vectors are unofficial according to thenomenclature rule of the ascidian community (Stolfi et al., 2015). Their officialnames are shown in our database.)
The unification of the backbone means that you can use the identical method tomodify our vectors. Please assume that you create the vector expressing eGFP in themuscle with the cis element of Troponin I (TnI) and NBRP pSPeGFP vector. When
you wish to create another vector expressing Kaede in the muscle with the same ciselement, you can use the same primer set, or PCR fragment, of TnI and pSPKaede tocreate a TnI>Kaede construct. The unified system saves time and money.
1) ORF switching
When you wish to replace the ORF of the pSP vector, you can digest a pSPxxxx
vector with EcoRI and either BamHI or NotI, to remove xxxx. The recommended
choice is NotI and EcoRI, because we can use the BamHI site to subclone ciselements (if the BamHI site is not present in the new ORF), and indeed wefrequently use BamHI to subclone cis elements. You may select other restriction
sites in the MCS to insert the ORF; however, please keep in mind that selectingother restriction sites will delete the BamHI site, which is frequently used to insert
cis elements.
The new ORF can be PCR-amplified. Traditionally PCR-amplified ORFs aredesigned to have BamHI/NotI and EcoRI sites at their ends and are inserted into theBamHI/NotI-EcoRI site of the pSP vector with a DNA ligase. Recently, the In-fusion system has been useful for the subcloning because the system is accurate andhighly efficient. Therefore, we recommend using the kit.
In-fusion requires 15 base homology arms at each end of the PCR fragment. You
can design the primers at the website (https://www.takarabio.co.jp/infusion_primer/infusion_primer_form.php). When an ORF is to beinserted into the NotI-EcoRI of pSPxxxx, the primer design of the new ORF will be
as follows:
GATCCCCTTGCGGCCGCTATGxxxxxxxxxxxxxx

CGCTCAGCTGGAATTxxxxxxxxxxxxxxxxxxxx

The xs corresponds to the new ORF. Homology arms are underlined. Red ATG is thestart codon of the new ORF.
2) Cis element switching
Cis elements can be amplified by PCR from the Ciona genome or from vectors thathave the cis elements of your interest. We usually linearize pSP vectors with BamHI
or NotI to insert a cis element. The In-fusion system is useful in this case, too. TheIn-fusion−compatible primers for inserting a cis element into the BamHI site of apSP vector are as follows:
CGACTCTAGAGGATCxxxxxxxxxxxxxxxxxx
GGCCGCAAGGGGATCxxxxxxxxxxxxxxxxxx
If you would like to add the start codon of the endogenous gene, the 3’ side primer isas follows:
GGCCGCAAGGGGATCCATxxxxxxxxxxxxxxxxxx
Red indicates the start codon.
If the pSPxxxx vector obeys our recommendation in section 1-1), you can re-use thePCR fragment of the cis element to subclone it into the BamHI of the pSPxxxx
vector by In-fusion system, because the homology arm matches. If the pSPxxxx
vector does not obey the rule, you may have to order another 3’ side primer for thesubcloning.
When isolating a new cis element whose activity has not been validated in theliterature or in experiments, we usually start by amplifying an approximately 2,000
bp fragment upstream from the start codon. Because the start codon usually presents
in the first exon, the fragment includes the region encoding the 5’UTR. When thedistance to the next gene is shorter than 2,000 bp, the length of the amplicon you use
should be reduced.
Tips: Some cis elements require longer upstream regions. If a new cis element does
not work, please isolate the longer fragment. In our experience, some enhancers areincluded in the first intron. In this case, the addition of the first intron would
improve the situation. The requirement of the first intron could be predicted by thehomology between C. intestinalis and C. savignyi (which can be found in the JGICiona genome browser). In the next section, we describe how to introduce
enhancers in expression vectors.
3) Introduction of enhancers
Sometimes we wish to introduce another DNA fragment in your pSP-cis
element>xxxx vector. There are two choices for the position of another DNA:
upstream of the cis element or downstream of the ORFs.
If the region upstream from the cis element is a good position, you can use arestriction site such as XhoI, SalI, or PstI for this purpose (Figure 1). Linearize
vectors with one of these restriction sites and insert the enhancer by In-fusion
reaction. This method is good when enhancers are insensitive to the position relative
to the gene or when you wish to insert a cis element that is too long to isolate by asingle PCR reaction.
When the first intron is necessary, we usually insert the DNA encoding the intron at
the downstream region of the ORF. For this purpose, the BglII site that is at the 3’
end of the transcription termination sequence (Ter) can be used.
In both cases, sometimes the restriction enzymes cannot be used because of thepresence of another restriction site in the inserted cis element or ORF. In such cases,
you can linearize the vector by inverse PCR.

Figure 1
EcoRI
Structure of pSP vectors

Transcription termination sequence (Ter)
BglII

Multicloning site (MCS)

ORF (xxxx)

Ter

Ampicillin-resistant gene

MCS of pSPnEGFP:

Start codon of NLS-eGFPSphICTCGAGCAGCTGAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTGCGGCCGCAAGGGTACGGCGCTCAGAAAAAatg
XhoI

HindIII

PstISalIXbaI

BamHI

NotI

The MCS sequence is not always identical between vectors because some nucleotides are lost during cloning; however, the reading frame isalways the same with respect to the restriction sites. For example, all vectors have 3n+1 nucleotides between the BamHI site and the start codon.