Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors

m834

tcatggtcatagctgtttcctg

pUC118 amplification, forward

m835

aacgtcgtgactgggaaaac

pUC118 amplification, reverse

m911

GGAAACAGCTATGACCATGAtcgaacggaagatcacttcgcag

CAT insert, forward

m912

GTTTTCCCAGTCACGACGTTgggcaccaataactgcctta

CAT insert, reverse

m915

ggcaatgaaagacggtgagc

CAT insert partial fragment 2, forward

k071

aacactatcccatatcaccagctcaccg

CAT insert partial fragment 1, reverse

k072

taagcagacagttttattgtgggcaccaataactgcctta

CAT insert partial fragment 2, reverse

k073

taaggcagttattggtgcccacaataaaactgtctgctta

Kan resistance insert partial fragment 1, forward

k074

caaccaaaccgttattcattcgtgattgcgcctgagcgag

Kan resistance insert partial fragment 1, reverse

k075

ctcgctcaggcgcaatcacgaatgaataacggtttggttg

Kan resistance insert partial fragment 2, forward

k076

ttaagttgggtaacgccagggttttcccagtcacgacgttcaggtggcacttttcggggaaatg

Kan resistance insert partial fragment 2, reverse

k089

gtttaactttaagaaggagatatacatatgCATCACCATCATCATCATtccgata

acgctcaacttaccggtc

ExoT cloning, forward

k090

ctttcgggctttgttagcagccggatccttacacctcttcggcggcagatag

ExoT cloning, reverse

k095

acccggggatcctctagagtcgacCTGCAGacggaagatcacttcgcag

CAT insert forward for PstI-site cloning, forward

k096

acggccagtgccaagcttgcatgcCTGCAGgggcaccaataactgcctta

CAT insert forward for PstI-site cloning, reverse

k261

ATTATAAAAATTAAAAAAATcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k262

ATTTTTTTAATTTTTATAATgggcaccaataactgcctta

Fig.3, supplemental Table S2

k263

AAATTTTTATATATTTTAAAcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k264

TTTAAAATATATAAAAATTTgggcaccaataactgcctta

Fig.3, supplemental Table S2

k265

TAATATTAGAAACTTATTATcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k266

ATAATAAGTTTCTAATATTAgggcaccaataactgcctta

Fig.3, supplemental Table S2

k267

TATAATAAGATGTAAATTAAcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k268

TTAATTTACATCTTATTATAgggcaccaataactgcctta

Fig.3, supplemental Table S2

k269

AATTTCAGATTCTTCTTATAcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k270

TATAAGAAGAATCTGAAATTgggcaccaataactgcctta

Fig.3, supplemental Table S2

k271

TATAACGTTCATTAAATGATcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k272

ATCATTTAATGAACGTTATAgggcaccaataactgcctta

Fig.3, supplemental Table S2

k273

GATTAAGTGAATTGACATGAcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k274

TCATGTCAATTCACTTAATCgggcaccaataactgcctta

Fig.3, supplemental Table S2

k275

CTGTTAAACAGACATACTAAcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k276

TTAGTATGTCTGTTTAACAGgggcaccaataactgcctta

Fig.3, supplemental Table S2

k277

CTCTAGTATACAGACGAGATcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k278

ATCTCGTCTGTATACTAGAGgggcaccaataactgcctta

Fig.3, supplemental Table S2

k279

TGCCTAATACCTCTCAAATGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k280

CATTTGAGAGGTATTAGGCAgggcaccaataactgcctta

Fig.3, supplemental Table S2

k281

TCCCCTTTACCACATGCTAGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k282

CTAGCATGTGGTAAGGGGAgggcaccaataactgcctta

Fig.3, supplemental Table S2

k283

ACGAGTCTCGATTTGCACTTcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k284

AAGTGCAAATCGAGACTCGTgggcaccaataactgcctta

Fig.3, supplemental Table S2

k285

CCCGACAAGTTAGCAGACCGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k286

CGGTCTGCTAACTTGTCGGGgggcaccaataactgcctta

Fig.3, supplemental Table S2

k287

AACACACGCAGTCTCCGGTGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k288

CACCGGAGACTGCGTGTGTTgggcaccaataactgcctta

Fig.3, supplemental Table S2

k289

GTCCGATCAGTGCCGGAGCCcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k290

GGCTCCGGCACTGATCGGACgggcaccaataactgcctta

Fig.3, supplemental Table S2

k291

GCCCTGAACCGCCCAACCCGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k292

CGGGTTGGGCGGTTCAGGGCgggcaccaataactgcctta

Fig.3, supplemental Table S2

k293

TGTGGGGAGGGGCTGCGCTCcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k294

GAGCGCAGCCCCTCCCCACAgggcaccaataactgcctta

Fig.3, supplemental Table S2

k295

GCTACGTGGCGCCCACGGCCcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k296

GGCCGTGGGCGCCACGTAGCgggcaccaataactgcctta

Fig.3, supplemental Table S2

k297

GGGGTCGCCGGTCCCGCCCCcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k298

GGGGCGGGACCGGCGACCCCgggcaccaataactgcctta

Fig.3, supplemental Table S2

k299

GCCCCCGGCGGCGTGGGGGAcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k300

TCCCCCACGCCGCCGGGGGCgggcaccaataactgcctta

Fig.3, supplemental Table S2

k301

CGGCGCCGGCCGCGCGCGCGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k302

CGCGCGCGCGGCCGGCGCCGgggcaccaataactgcctta

Fig.3, supplemental Table S2

k303

GCCGGCGCGCCCCCCGCCGGcctggcgttacccaacttaa

Fig.3, supplemental Table S2

k304

CCGGCGGGGGGCGCGCCGGCgggcaccaataactgcctta

Fig.3, supplemental Table S2

Supplemental Table S1

Primers used in the present study. All primers were synthesized by Eurofins on a

scale of 10 nmoles and purified using the OPC protocol.

experiment

GC%

vector number

vector primer

insert number

insert primer

V1

m834,k261

I1

m911,k262

V2

m834,k263

I2

m911,k264

10

V3

m834,k265

I3

m911,k266

10

V4

m834,k267

I4

m911,k268

20

V5

m834,k269

I5

m911,k270

20

V6

m834,k271

I6

m911,k272

30

V7

m834,k273

I7

m911,k274

30

V8

m834,k275

I8

m911,k276

40

V9

m834,k277

I9

m911,k278

10

40

V10

m834,k279

I10

m911,k280

11

50

V11

m834,k281

I11

m911,k282

12

50

V12

m834,k283

I12

m911,k284

13

60

V13

m834,k285

I13

m911,k286

14

60

V14

m834,k287

I14

m911,k288

15

70

V15

m834,k289

I15

m911,k290

16

70

V16

m834,k291

I16

m911,k292

17

80

V17

m834,k293

I17

m911,k294

18

80

V18

m834,k295

I18

m911,k296

19

90

V19

m834,k297

I19

m911,k298

20

90

V20

m834,k299

I20

m911,k300

21

100

V21

m834,k301

I21

m911,k302

22

100

V22

m834,k303

I22

m911,k304

Supplemental Table S2

Design for the amplification of DNA fragments used in Fig. 3. Twenty-two pairs of

vectors and insert fragments were generated and used in experiments with 22

different homology arm sequences. Each experiment was repeated at least three

times, and the data obtained are shown in Fig. 3.

Supplemental Figures and Tables, legends

Figure S1

Schematic diagram of two types of seamless DNA cloning

In seamless DNA cloning, such as Gibson Assembly and In-Fusion HD Cloning, 15- to 20-bp

homology arm sequences are designed and generated by PCR at both ends of the insert fragment. The

homology arm region may essentially be any sequence as long as the common sequence appears at the

end of the insert and vector. One pair of an insert and vector is shown. Depending on the enzyme used

in the kit, a 3’->5’ (left panel) or 5’->3’ (right panel) resection is generated. The ssDNA region

generated in the homology arm of the insert is complementary to that in the vector. These strands

spontaneously form dsDNA, which is repaired by other enzymes in the reaction or in E. coli cells after

transformation.

Figure S2

Evaluation of the ChlR insert cloned by the XE cocktail.

(a) Schematic diagram of cloning followed by the replica plating experiment. The ChlR insert was

amplified and cloned into a linearized ampicillin-resistant plasmid. The resulting transformants were

then plated onto LB-ampicillin plates. One hundred colonies were then randomly selected and

inoculated on LB-chloramphenicol plates.

(b) The numbers of chloramphenicol-resistant and -sensitive colonies were counted and indicated.

Three independent assembly reactions were performed and examined separately.

Table S1

Primers used in the present study. All primers were synthesized by Eurofins on a scale of 10 nmoles

and purified using the OPC protocol.

Table S2

Design for the amplification of DNA fragments used in Fig. 3. Twenty-two pairs of vectors and insert

fragments were generated and used in experiments with 22 different homology arm sequences. Each

experiment was repeated at least three times, and the data obtained are shown in Fig. 3.

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