1. Methods S2. Detailed pDOE vector schematics and cloning strategies.
( pg. 1 )
BiFC vector sets
MCS1
MCS3
MCS2
p19/XT-mTq2
MASpro
Pro::Ω
NmVen210
OCSt
CVen210
MASt
NOSt
X::tag—X::tag
pDOE-01, -05, -09
X::tag—tag::X
pDOE-02, -06, -10
tag::X—X::tag
pDOE-03, -07, -11
tag::X—tag::X
pDOE-04, -08, -12
Fluoroprotein fusion vector sets
MCS1
MCS3
MCS2
p19
MASpro
Pro::Ω
mTurquoise2
OCSt
mVenus
MASt
NOSt
X::tag—X::tag
pDOE-13, -17
X::tag—tag::X
pDOE-14, -18
tag::X—X::tag
pDOE-15, -19
tag::X—tag::X
pDOE-16, -20

2. ( pg. 2 )
Single sheet composite view of all four pDOE multiple cloning site arrangements
This page provides the restriction enzyme recognition site sequences and their relationship to the open reading frame
Restriction sites are directly above the enzyme name and the trailing underscore. (e.g. NcoI__ = ccATGG)
MCS1 X-tag:
M G R A L G T S G G S G G G S G S R P G G . L
ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NVen210]tctagacccgggggttaattaa
NcoI__AscI____ SpeI__ BamHI_ XbaI__SmaI__ PacI____
AvrII_ BspEI_(dam dead)
MCS1 tag-X:
G A S G G G S G S M G R A L G T S . S R P G G . L
[NVen210]GGAGCCAGTGGAGGTGGGTCAGGATCCATGGGGCGCGCCCTAGGAACTAGTtaatctagacccgggggttaattaa
NcoI_ AscI____ SpeI_ XbaI__SmaI__ PacI____
BamHI_ AvrII_
MCS3 X-tag:
M G S L R S H V T S G G S G G A A I A I .
caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat
SanDI__SnaBI_ Eco721 BspEI_ AsiSI___ SwaI____
AatII_
MCS3 tag-X:
S G A S G M G S L R S H V T S G . A I A I . I
[CVen210-SFS]TCTGGAGCTTCAGGAATGGGGTCCCTACGTAGTCACGTGACGTCCGGAtaagcgatcgccatttaaat
SanDI__SnaBI_ Eco721 BspEI_ AsiSI___ SwaI____
Deviation:
In the “FRET” vectors the
last Ala in the linker is Gly
for X::mVenus
Deviation:
In the “FRET” vectors the
mVenus::X constructs have a longer linker
(SGAGSGGGSGM) and
the stop codon is TGA

3. Comprehensive overview of the individual BiFC systems
( pg. 3 )
MCS1 (X::tag)
M G R A L G T S G G S G G G S G S R P G G . L
ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa
NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI
MCS3 (X::tag)
M G S L R S H V T S G G S G G A A I A I .
caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat
SanDI SnaBI PmlI AatII BspEI AsiSI SwaI
MCS1
MCS3
MCS2
p19/XT-mTq2
MASpro
Pro::Ω
NmVen210
OCSt
CVen210
MASt
NOSt
KpnI
NruI
XhoI
Bsu36I
*BstXI
EcoRI
1) X::tag—X::tag
pDOE-01, -05, -09
MCS1 (X::tag)
M G R A L G T S G G S G G G S G S R P G G . L
ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa
NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI
MCS3 (tag::X)
S G A S G M G S L R S H V T S G . A I A I . I
[CVen210-SFS]TCTGGAGCTTCAGGAATGGGGTCCCTACGTAGTCACGTGACGTCCGGAtaagcgatcgccatttaaat
SanDI SnaBI PmlI AatII BspEI AsiSI SwaI
MCS1
MCS2
MCS3
KpnI
NruI
p19/XT-mTq2
MASpro
NmVen210
OCSt
MASt
CVen210
NOSt
Pro::Ω
XhoI
Bsu36I
*BstXI
EcoRI
2) X::tag—tag::X
pDOE-02, -06, -10

4. ( pg. 4 ) 3) tag::X—X::tag pDOE-03, -07, -11 4) tag::X—tag::X
MCS1 (tag::X)
G A S G G G S G S M G R A L G T S . S R P G G . L
[NVen210]GGAGCCAGTGGAGGTGGGTCAGGATCCATGGGGCGCGCCCTAGGAACTAGTtaatctagacccgggggttaattaa
BamHI NcoI AscI AvrII SpeI XbaI SmaI PacI
MCS3 (X::tag)
M G S L R S H V T S G G S G G A A I A I .
caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat
SanDI SnaBI PmlI AatII BspEI AsiSI SwaI
MCS1
MCS2
XhoI
MCS3
KpnI
NruI
p19/XT-mTq2
MASpro
NmVen210
OCSt
MASt
CVen210
NOSt
Pro::Ω
Bsu36I
*BstXI
EcoRI
3) tag::X—X::tag
pDOE-03, -07, -11
MCS1 (tag::X)
G A S G G G S G S M G R A L G T S . S R P G G . L
[NVen210]GGAGCCAGTGGAGGTGGGTCAGGATCCATGGGGCGCGCCCTAGGAACTAGTtaatctagacccgggggttaattaa
BamHI NcoI AscI AvrII SpeI XbaI SmaI PacI
MCS3 (tag::X)
S G A S G M G S L R S H V T S G . A I A I . I
[CVen210-SFS]TCTGGAGCTTCAGGAATGGGGTCCCTACGTAGTCACGTGACGTCCGGAtaagcgatcgccatttaaat
SanDI SnaBI PmlI AatII BspEI AsiSI SwaI
MCS1
MCS2
KpnI
NruI
Bsu36I
MCS3
p19/XT-mTq2
MASpro
NmVen210
OCSt
CVen210
MASt
NOSt
Pro::Ω
XhoI
*BstXI
EcoRI
4) tag::X—tag::X
pDOE-04, -08, -12

5. Cloning options for MCS1 BamHI = ..ATCC (Ser) BclI = ..ATCA (Ser)
( pg. 5 )
Cloning options
for MCS1
BamHI = ..ATCC (Ser)
BclI = ..ATCA (Ser)
BglII = ..ATCT (Ser)
MCS1 (X::tag)
M G R A L G T S G G S G G G S G S R P G G . L
ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa
NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI
Insert PCR product cut with:
NcoI = ..ATGG
BspHI = ..ATGA
PciI = ..ATGT
If your ORF is ..ATGC, insert a novel 2nd codon starting
with G,A, or T such as Gly (GGN), e.g. ATG GGN [CNN]
Tag removal and other conjugations via compatible ends:
AvrII
SpeI
XbaI
in frame
NEB BspEI will not cut.
ThermoFisher BspEI will cut this site
and the site in MCS3; religating creates
a MCS1/3 hybrid for tag exchange, single
expression, etc.
Additional options:
1) Erase the NcoI site in the ORF by overlap PCR.
This is a better option overall, and allows for easy retrieval of the ORF.
2) Utilize the rare cutting AscI site.

6. Cloning options for MCS3 AatII out-of-frame cohesive cut
( pg.6 )
Cloning options
for MCS3
AatII out-of-frame cohesive cut
ZraI out-of-frame blunt cut
MCS3 (X::tag)
M G S L R S H V T S G G S G G A A I A I .
caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat
SanDI SnaBI PmlI AatII BspEI AsiSI SwaI
PCR product contribution
underlined:
SanDI = GGGTCCC
RsrII = CGGTCCG
AvaII = GGTCCn
PpuMI = RGGTCCY
PmlI/Eco72I
in-frame blunt cut
BspEI compatible sites:
AgeI/BshTI = ACCGGT
XmaI = CCCGGG
NgoMIV = GCCGGC
(plus others*)
*BsaWI (WCCGGW)
BsrFI (RCCGGY)
If the ORF for your gene is M[X] where x = L, R, P, H, Q your primer is ATG GGG TCC [X] = MGS[X] with a SanDI site
If the ORF for your gene is M[X] where x = G, A, V, E, D your primer is ATG CGG TCC [X] = MRS[X] with an RsrII site
RG after ligation
If the second residue of your ORF is not one of the ten residues noted above, choose one to add as a third inserted codon, e.g. MGSG[X]…
* For SanDI, your primer will be ATG GGG TCC CNN [native ORF 2nd residue]
* For RsrII, your primer will be ATG CGG TCC GNN [native ORF 2nd residue]

7. Constructions are made in the same vector,
( pg. 7 )
Constructions are made in the same vector,
and the pDOE system can be thought of as one large MCS.
For example:
1) Cloning into NcoI + an MCS3 upstream site yields a single cassette ORF::CVen210 construct (e.g. NcoI + MCS3 BspEI)
— Orientations besides the one shown will give other products
2) Cloning into NcoI + AsiSI yields a single cassette untagged protein
3) Ligating the blunt cut sites of SmaI + SwaI deletes the third cassette (and tails MCS1 with the NOSt)
MCS1 (X::tag)
M G R A L G T S G G S G G G S G S R P G G . L
ccATGGGGCGCGCCCTAGGAACTAGTGGAGGATCCGGAGGTGGGTCAGGA[NmVen210]tctagacccgggggttaattaa
NcoI AscI AvrII SpeI BamHI BspEI (methyl blocked) XbaI SmaI PacI
BspEI MCS1/3 merge also allows fluoroprotein exhange:
Cloned MCS1 ORF::mTq2  cut/religate = ORF::mVenus
This merge can also doubly fluorotag a protein:
Cloned MCS1 mTq2 ORF  cut/religate = mTq2::ORF::mVenus
MCS3 (X::tag)
M G S L R S H V T S G G S G G A A I A I .
caATGGGGTCCCTACGTAGTCACGTGACGTCCGGAGGTTCTGGTGGAGCT[SFS-CVen210]gcgatcgccatttaaat
SanDI SnaBI PmlI AatII BspEI AsiSI SwaI