Construction of expression vectors for Fusion proteins
Yasunori Sasakura 15th December 2017
Modified 26th January 20231) Conventional fusions
In order to express a fusion protein of two ORFs, we usually generate an expression
vector by the following procedure:
Insert the N-terminal side ORF into the BamHI or NotI and EcoRI of the pSP vector.
EcoRI must remain after the insertion. The stop codon must be deleted from theORF. Then insert the C-terminal side ORF at the EcoRI site (by means of ligation orIn-fusion).
NBRP provides pSPCFP-ter. The vector was used to make the eCFP::Hox1 fusion
construct (Sasakura et al., 2012 Development 139, 2156-2160).
2) Using 2A peptide
Recently, the 2A peptide sequence was used in Ciona. This sequence allows us toexpress two ORFs from a single cis element without making a fusion of the twoORFs. This system can minimize the modifications to ORFs and subsequently
would reduce the risk of impairing the activities of proteins by fusing to something.
We have established a systematic way to create 2A fusion expression vectors (e.g.
Krasovec et al., 2022, Sci. Adv. 8, eabn3264). The vector designs are shown in
Figure 1 (ORF::2A::FP) and Figure 2 (FP::2A::ORF). FP means fluorescent protein.
2-1) making ORF::2A::FP constructs (Figure 1)
Insert your ORF at the NotI site of a pSP vector compatible with the 2A system(such as pSP2AeGFP; Figure 1). The primers for amplifying the ORF will be asfollows:
GATCCCCTTGCGGCCATGxxxxxxx
CCTGATCCTGCGGCCGCxxxxxxxx (Please delete the stopcodon.)The red ATG is the start codon of your ORF. The nucleotide length of the amplified

ORFs should be a multiple of 3 (in order to adjust the reading frame). For thisreason, the red x must be at the +3 position of the codon. You must delete the stopcodon from the amplified ORF. You can insert promoters upstream from the ORF
using a restriction site. When you use the BamHI site, you can re-use the promoter
fragment used in section 1. When a BamHI site is present in your ORF, you can first
insert the promoter and then insert the ORF, or you can open the vector by inversePCR.
2-2) making FP::2A::ORF constructs (Figure 2)
Insert your ORF at the EcoRI site of a pSP vector compatible with the 2A system(such as pSPeGFP2A; Figure 2). The primers for amplifying the ORF will be asfollows:
CCCAGGACCAGAATTCxxxxxxxxxx (Please delete the startcodon.)
CGCTCAGCTGGAATTCTTAxxxxxxx
The red TTA corresponds to the stop codon (you can change the type of stop codon).
The nucleotide length of the amplified ORFs should be a multiple of 3 (in order toadjust the reading frame). The red x must be the +1 of the codon. We usually delete
the start codon from the amplified ORF to make sure that the ATG is used at the firstORF. You can remove the inserted ORF and insert a new ORF with EcoRI.
You can insert promoters in the MCS. The BamHI site is compatible with other pSP
vector systems, and you can use the primer sets mentioned section 1-2. When aBamHI site is present in your ORF, please first insert the promoter and then insert
the ORF, or you can open the FP::2A::ORF construct by inverse PCR.

Figure 1
Structure of the pSP-2A peptide vector for fusing an ORF at the N-terminal side of a fluorescent protein(ORF::2A::FP)EcoRI
2A peptide sequence
MCS

MCS of pSPORF::2A::FP
SphI

BglII
2A

FP (e.g., GFP)Ter

Start(+1) of FP ORF

CTCGAGCAGCTGAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTGCGGCCGCAggatcaggagaa.....caggaccaGTGAGCAAG
Red, 2A peptide (63 nt long)
XhoI
HindIII
PstI SalI XbaI BamHI
NotI

Figure 2
Structure of the pSP vector for fusing an ORF at the C-terminal of a fluorescent protein(FP::2A::ORF)

EcoRI

BglII

Multicloning site (MCS)FP (e.g., GFP)

2A

Ter

2A peptide sequence
MCS of pSPFP::2A::ORF

Start codon of FP

SphI

CTCGAGCAGCTGAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTTGCGGCCGCTatg
XhoI

HindIII

PstI

SalI

XbaI

BamHI

NotI

Around the EcoRI site
EcoRI Termination sequence
aagGGATCAGGAGAAGGAAGAGGATCACTTCTTACATGTGGAGATGTTGAAGAAAACCCAGGACCAgaattcCAGCTGAGCGCCGGTCGCTAC...
Red, 2A peptide (63 nt long)
The end (+3) of FP (in this case, eGFP)