整合酶重組系統讓載體構建像搭積木一樣簡單高效

1234—Entry

clonecDNADNA……PCRNGatewayGateway

Thermo

FisherBPCat:

SBBP011LRCat:SBLR01…1N…BPCat:

SBBP01P1

P2ccdBB1

B2GOIattB

PCR

產物+L1

L2TOPODonor

VectorL1

L2Digested

Entry

VectorL1

L2Digested

Entry

VectorGOILigaseGOITopo位點PCR

產物+TOPOBPTopo-T4Gibson

assembly/NEBuilderIn-fusionGOI+DNA

LR酶B1B2B5GOI

1GOI

2骨架載體LR酶（Cat:SBLR01）-B1-GOI1-B5r-BP酶+-P1-ccdB-P5r--B5-GOI2-B2-+-P5-ccdB-P2-BP酶BPR7種B位點/(PCR/

)Donor(

)(

/

)1B1-B2P1-P2R1-R2B1-GOI1-B2103-1062B1-B5r,

B5-B2P1-P5r,

P5-P2B1-GOI1-B5r-GOI2-B2103-1053B1-B4,B4r-B3r,B3-B2P1-P4,P4r-P3r,P3-P2B1-GOI1-B4-GOI2-B3r-GOI3-B2103-1044B1-B5r,

B5-B4,B4r-B3r,B3-B2P1-P5r,P5-P4,P4r-P3r,P3-P2B1-GOI1-B5r-GOI2-B4-GOI3-B3r-GOI4-B2102-1045B1-B4,B4r-B3r,B3-B6,B6r-B5r,B5-B2P1-P4,P4r-P3r,P3-P6,P6r-P5r,P5-P2B1-GOI1-B4-GOI2-B3r-GOI3-B6-GOI4-B5r-GOI5-B2101-102更多B5-GOIX-B4P5-P4-GOI-R5-R4-GOI-B1-GOI-B5r-GOIX-B4-GOI-B3r-GOI-B2/

17,963

Gateway?

Cloning

Vectorshttps://ww/plasmids/gateway_cloning_vectors10610+

https:///tutorials/topo-cloning

—Golden

Gate

AssemblySnapgeneType

IIPGolden

Gate

AssemblyType

IISBsaI

1.2.3.Fidelity

vs.

Complexity

of

Golden

Gate

Assemblyhttps:///zh-cn/golden-gate/golden-gateNEBuilder是NEB的商標、GeneArt是ThermoFisher的商標、In-Fusion是Takara的商標

One-step—12DNA…

…—

One-stepPCRLR——PCR

————1234——

10

fmol/30-200

fmol96-wellClickWayGibson/In-fusion—None56~640.8/

35-40

nt/None10~30

(∞)None0.5~1PCRNone3~20None5None1None6~45None3~5NoneNone6~1850

μLNone30~50None10035~960.1-396-well3~50.50.10.1None40~4002

kb

1 10

/5

kb

4 10

/None2None0.5~1100146~6430.1-380~132<

12.1~3.1ClickWay/TopopENTR/D-TOPO/-20℃BPGateway

BP

ClonaseBPAB-80℃

/

6GatewayBP

Clonase

IIBP-20℃

/

6ClickWay

BPBP-80℃

/

12-20℃

/

8LRGateway

LR

ClonaseLRAB-80℃

/

6Gateway

LR

Clonase

IILR-20℃

/

6LR

Clonase

II

plusLR-80℃

/

6ClickWay

LR/

LRAB-80℃

/

12-20℃

/

8pENTR

TOPO

Gateway

Clonase

Thermo

FisherClickWay

LR

4-

LR1/3

-20℃1~

-80℃100%3~100%

?——DNA——Cat:NE001?——37℃

3LRClickWayGateway

(II

Plus)LR(

SBLR01)-80℃

1-20℃

8-80℃

(>10-20℃GateWay

LR

clonase

II-20℃

6-80℃

6LR

clonase

II

plusBPP1-P2Donor??pDV-P1P2Q4

—E.

coli

High

Yield2~

∞EcHiYi LREcHiYi8/24Q3Q4

RNAi(CRISPR)…

…

CRISPR…

…RNAi…

…

Gateway

clonase

Thermo

FisherClickWayClickWayMultiSite

Gateway

Kit(Addgene#1000000050)?NCL1-[ORF]-L2L4-[N-module]-R1R2-[N-module]-L3R4-ccdB-R3……Raquel

Buj,

et

al.

BMC

Mol

Biol.

2013.14:18.EcHiYiEcHiYiEcHiYi5

~

∞EcHiYi—Yao

Xiao

and

Remko

Offringa.

Nat

Plants.

2020Michniewicz

et

al.

BMC

Res

Notes.

2015

PDK1/PDK2

Promoter::GFP:GUSGFP:GUSCRISPR-Cas9

knockoutgRNA

LR

CRISPRpCambia-pYAO-Cas9-

gRNA1/2/3

pART7-35S::YFP:PDK1FL/S0/S1/S2pMDC32-35S::PDK1FL/S0/S1/S2

pGEX-PDK1FL/S0pGEX-PIN1HLpGEX-PIN2HLpGEX-PAXLR:attR1attR2ccdBPromoterGOIattL1attL2LRMichniewicz

et

al.

BMC

Res

Notes.

2015ID25767pEN_TRE_GFPEntry

vector

with

TRE

promoter

driving

GFP

expression.25766pEN_TGmiR_Gb2-KEntry

vector

withTRE

promoter

driving

mouse

G

beta

2

miR30-based

shRNA

with

GFP

coexpression.25765pEN_TGmiR_Luc-AEntry

vector

withTRE

promoter

driving

control

Luciferase

miR30-based

shRNA

with

GFP

coexpression.25764pEN_TmiR_Gb2-KEntry

vector

with

TRE

promoter

driving

mouse

G

beta

2

miR30-based

shRNA.25763pEN_TmiR_G13-L_G12-EEntry

vector

with

TRE

promoter

driving

both

mouse

G

alpha

12

and

G

alpha

13

miR30-based

shRNAs.25762pEN_TmiR_G13-LEntry

vector

with

TRE

promoter

driving

mouse

G

alpha

13

miR30-based

shRNA.25761pEN_TmiR_G12-EEntry

vector

with

TRE

promoter

driving

mouse

G

alpha

12

miR30-based

shRNA.25760pEN_TmiR_Luc-AEntry

vector

withTRE

promoter

driving

control

Luciferase

miR30-based

shRNA.25759pEN_hU6miR-Gb2-KEntry

vector

with

human

U6

promoter

driving

both

mouse

G

alpha

12

and

G

alpha

13

miR30-based

shRNAs.25758pEN_hU6miR-G13-LEntry

vector

with

human

U6

promoter

driving

mouse

G

alpha

13

miR30-based

shRNA.25757pEN_hU6miR-G12-EEntry

vector

with

human

U6

promoter

driving

mouse

G

alpha

12

miR30-based

shRNA.25756pEN_hU6miR-Luc-AEntry

vector

with

human

U6

promoter

driving

control

Luciferase

miR30-based

shRNA.25755pEN_TTmcsEntryvector

withTRE-tight

promoter

followed

by

multiple

cloningsite.25754pEN_TTRmiRc2Entry

vector

for

cloning

miR30-based

shRNA

driven

by

TRE-tight

promoter

with

dsRed2

coexpression.25753pEN_TTGmiRc2Entry

vector

for

cloning

miR30-based

shRNA

driven

by

TRE-tight

promoter

with

GFP

coexpression.25752pEN_TTmiRc2Entry

vector

for

cloning

miR30-based

shRNA

driven

by

TRE-tight

promoter.25751pEN_TmcsEntry

vector

with

TRE

promoter

followed

by

multiple

cloning

site.25750pEN_TRmiRc2Entry

vector

for

cloning

miR30-based

shRNA

driven

by

TRE

promoter

with

dsRed2

coexpression.25749pEN_TGmiRc3Entry

vector

for

cloning

miR30-based

shRNA

driven

by

TRE

promoter

with

GFP

coexpression.25748pEN_TmiRc3Entry

vector

for

cloning

miR30-based

shRNA

driven

by

TRE

promoter.25747pEN_hUmiRc2Entry

vector

for

cloning

miR30-based

shRNA

driven

by

human

U6

promoter.25746pSLIK-Neo-TGmiR-Gb2Control

lentiviral

vector

with

Tet-based

inducible

expression

of

mouse

G

beta

2

miR30-based

shRNA

and

GFP,

constitutive

Neomycin

resistance

gene

coexpression.25745pSLIK-Neo-TGmiR-LucControl

lentiviral

vector

with

Tet-based

inducible

expression

of

Luciferase

miR30-based

shRNA

and

GFP,

constitutive

Neomycin

resistance

gene

coexpression.25744pSLIK-Neo-TmiR-Gb2Control

lentiviral

vector

with

Tet-based

inducible

expression

of

mouse

G

beta

2

miR30-based

shRNA,

constitutive

Neomycin

resistance

gene

coexpression.25743pSLIK-Neo-TmiR-LucControl

lentiviral

vector

with

Tet-based

inducible

expression

of

Luciferase

miR30-based

shRNA,

constitutive

Neomycin

resistance

gene

coexpression.25742pSLIK-Venus-TmiR-G13-G12Lentiviral

vector

with

Tet-based

inducible

expression

of

both

mouse

G

alpha

12

and

G

alpha

13

miR30-based

shRNAs,

constitutive

Venus

fluorescent

protein

coexpression.25741pSLIK-Venus-TmiR-G13Lentiviral

vector

with

Tet-based

inducible

expression

of

mouse

G

alpha

13

miR30-based

shRNA,

constitutive

Venus

fluorescent

protein

coexpression.25740pSLIK-Venus-TmiR-G12Lentiviral

vector

with

Tet-based

inducible

expression

of

mouse

G

alpha

12

miR30-based

shRNA,

constitutive

Venus

fluorescent

protein

coexpression.25739pSLIK-Venus-TmiR-LucControl

lentiviral

vector

with

Tet-based

inducible

expression

of

Luciferase

miR30-based

shRNA,

constitutive

Venus

fluorescent

protein

coexpression.25737pSLIK-Hygro3rd

generation

Lentiviral

vector

for

Tet-based

inducible

shRNA

or

cDNA

expression,

constitutive

Hygromycin

resistance

gene

coexpression.25736pSLIK-ZeoLentiviral

vector

for

Tet-based

inducible

shRNA

or

cDNA

expression,

constitutive

Zeocin

resistance

gene

coexpression.25735pSLIK-Neo3rd

generation

Lentiviral

vector

for

Tet-based

inducible

shRNA

or

cDNA

expression,

constitutive

Neomycin

resistance

gene

coexpression.25734pSLIK-VenusLentiviral

vector

for

Tet-based

inducible

shRNA

or

cDNA

expression,

constitutive

Venus

fluorescent

protein

coexpression.21TRE12attR1attR2LRKum-Joo

Shin,

et

al.

PNAS.

2006.3N

to

1Multi-Knock——CRISPRCR