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Table 1 RSFC vector family designed for P. pastoris

From: Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

#

Name

Tag/Fusion protein, positiona and lengthb

TEV protease cleavage site

Mode of expression

EAEA repeat

Selection markerc

Plasmid size (bp)

1

PPpRSFC

–

–

–

NA

Intracellular

NA

Zeocin

4,840

2

pPpRSFC-MFalpha

–

–

–

NA

Secretory

Yes

Zeocin

5,104

3

pPpRSFC-MFalpha-noEAEA

–

–

–

NA

Secretory

No

Zeocin

5,092

4

pPpRSFC-pGAP

–

–

–

NA

Intracellular

NA

Zeocin

3,771

5

pPpRSFC-pGAP-MFalpha

–

–

–

NA

Secretory

Yes

Zeocin

4,035

6

pPpRSFC-pGAP-MFalpha-noEAEA

–

–

–

NA

Secretory

No

Zeocin

4,023

7

pPpRSFC-N-eGFP

eGFP

N

240

No

Intracellular

NA

Zeocin

5,584

8

pPpRSFC-C-eGFP

eGFP

C

239

No

Intracellular

NA

Zeocin

5,584

9

pPpRSFC-MFalpha-N-eGFP

eGFP

N

239

No

Secretory

Yes

Zeocin

5,848

10

pPpRSFC-MFalpha-C-eGFP

eGFP

C

239

No

Secretory

Yes

Zeocin

5,848

11

pPpRSFC-N-Myc

MYC

N

11

No

Intracellular

NA

Zeocin

4,870

12

pPpRSFC-C-Myc

MYC

C

10

No

Intracellular

NA

Zeocin

4,870

13

pPpRSFC-MFalpha-N-Myc

MYC

N

10

No

Secretory

Yes

Zeocin

5,134

14

pPpRSFC-MFalpha-C-Myc

MYC

C

10

No

Secretory

Yes

Zeocin

5,134

15

pPpRSFC-N-FLAG

FLAG

N

9

No

Intracellular

NA

Zeocin

4,864

16

pPpRSFC-C-FLAG

FLAG

C

8

No

Intracellular

NA

Zeocin

4,864

17

pPpRSFC-MFalpha-N-FLAG

FLAG

N

8

No

Secretory

Yes

Zeocin

5,128

18

pPpRSFC-MFalpha-C-FLAG

FLAG

C

8

No

Secretory

Yes

Zeocin

5,128

19

pPpRSFC-N-His-ncs

His

N

7

No

Intracellular

NA

Zeocin

4,858

20

pPpRSFC-C-His-ncs

His

C

6

No

Intracellular

NA

Zeocin

4,858

21

pPpRSFC-MFalpha-N-His-ncs

His

N

6

No

Secretory

Yes

Zeocin

5,122

22

pPpRSFC-MFalpha-C-His-ncs

His

C

6

No

Secretory

Yes

Zeocin

5,122

23

pPpRSFC-N-His

His

N

7

Yes

Intracellular

NA

Zeocin

4,879

24

pPpRSFC-C-His

His

C

6

Yes

Intracellular

NA

Zeocin

4,879

25

pPpRSFC-MFalpha-N-His

His

N

6

Yes

Secretory

Yes

Zeocin

5,143

26

pPpRSFC-MFalpha-C-His

His

C

6

Yes

Secretory

Yes

Zeocin

5,143

27

pPpRSFC-N-MBP

MBP

N

367

Yes

Intracellular

NA

Zeocin

5,959

28

pPpRSFC-C-MBP

MBP

C

366

Yes

Intracellular

NA

Zeocin

5,959

29

pPpRSFC-MFalpha-N-MBP

MBP

N

366

Yes

Secretory

Yes

Zeocin

6,223

30

pPpRSFC-MFalpha-C-MBP

MBP

C

366

Yes

Secretory

Yes

Zeocin

6,223

31

pPpRSFC-N-Strep

Strep

N

9

Yes

Intracellular

NA

Zeocin

4,885

32

pPpRSFC-C-Strep

Strep

C

8

Yes

Intracellular

NA

Zeocin

4,885

33

pPpRSFC-MFalpha-N-Strep

Strep

N

8

Yes

Secretory

Yes

Zeocin

5,149

34

pPpRSFC-MFalpha-C-Strep

Strep

C

8

Yes

Secretory

Yes

Zeocin

5,149

35

pPpRSFC-HIS

–

–

–

NA

Intracellular

NA

HIS4

7,683

36

pPpRSFC-HIS-MFalpha

–

–

–

NA

Secretory

Yes

HIS4

7,947

37

pPpRSFC-HIS-MFalpha-noEAEA

–

–

–

NA

Secretory

No

HIS4

7,935

38

pPpRSFC-HIS-pGAP

–

–

–

NA

Intracellular

NA

HIS4

6,614

39

pPpRSFC-HIS-pGAP-MFalpha

–

–

–

NA

Secretory

Yes

HIS4

6,878

40

pPpRSFC-HIS-pGAP-MFalpha-noEAEA

–

–

–

NA

Secretory

No

HIS4

6,866

  1. NA not applicable, ncs no TEV protease cleavage site.
  2. aN- or C- terminal fusion to the POI.
  3. bLength in amino acids (intracellular N- terminal tags are because of the start codon one aa longer, the TEV protease cleavage site (seven aa) is not included in this number).
  4. cZeocin selection is applicable for E. coli and P. pastoris, HIS4 plasmids are based on ampicillin selection in E. coli and used in combination with a histidine auxotrophic (his4) P. pastoris strain.