DNA Camouflage - DTIC · all sequencing reactions and QS/CRL measurements were performed by GENEWIZ...

1

DNA Camouflage

Supplementary Information

Bijan Zakeri1,2*, Timothy K. Lu1,2*, Peter A. Carr2,3*

1Department of Electrical Engineering and Computer Science, Department of Biological

Engineering, Research Laboratory of Electronics, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

2MIT Synthetic Biology Center, 500 Technology Square, Cambridge MA 02139, USA 3MIT Lincoln Laboratory, 244 Wood Street, Lexington MA 02420, USA

*Correspondence to Bijan Zakeri ([email protected]), Timothy K. Lu

([email protected]), and Peter A. Carr ([email protected]).

Distribution A: Public Release

2

Supplementary Figure 1 DNA camouflage with the 2-state device. (a) In the presence of Cre, DSD-2[α] was randomly shuffled between α and β states within a cellular population. (b) Quality score (QS) values of sequencing reactions of DSD-2[α] maintained in the absence and presence of Cre. (c) Contiguous read length (CRL) scores of sequencing reactions of DSD-2[α] maintained in the absence and presence of Cre. All experiments were performed in triplicate, error bars represent ± 1 standard deviation, and all sequencing reactions and QS/CRL measurements were performed by GENEWIZ Inc. under blind experimental conditions. QS scores below 24 and CRL scores below 500 indicate problems with sequencing results.

SI Fig. 1

a

b

c

Fig. 1. pBZ22 -/+ pBZ14

in vivo

in cognito

Induction (min) 120

0 60

120

Contiguous Read Length

Pass

0 250 500 750 1,000 1,250

in vivo

in cognito

Induction (min) 120

0 60

120

Quality Score 0 10 20 30 40 50

Pass

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 1,000 Length (bp)

Chromatogram Alignment

Template

0

60

120

Frequency (%) 0 50 100

α

α

α

β

β

β

in cognito

Induction (min)

0% 50% 100%

120$

60$

0$

alpha$

beta$

+ Cre α β


0 10 20 30 40 50

pBZ22 + pBZ14 - 120 min

pBZ22 + pET28a - 120 min

pBZ22%DSD2%QS%

Quality Score

− Cre

+ Cre

Pass

50 40 30 20 10 0

0 250 500 750 1000 1250

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%CRL%


− Cre

+ Cre

Pass

250 0 500 750 1000 1250

Fig. 1. pBZ22 -/+ pBZ14

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

0 250 500 750 1,000 1,250

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 1,000 Length (bp)


Template

0

60

120


α

α

α

β

β

β

in cognito

Induction (min)

0% 50% 100%

120$

60$

0$

alpha$

beta$

+ Cre α β


0 10 20 30 40 50

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%QS%

Quality Score

− Cre

+ Cre

Pass

50 40 30 20 10 0

0 250 500 750 1000 1250

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%CRL%


− Cre

+ Cre

Pass

250 0 500 750 1000 1250

3

Supplementary Figure 2 DNA shuffling does not comprise sequencing outside of DSDs. (a) Sequencing of 1 kb downstream of DSD-2[α] produces high quality sequencing reads that align with the template in the absence and presence of Cre. (b) Quality score (QS) and (c) Contiguous read length (CRL) scores of sequencing reactions shown in a. All experiments were performed in triplicate, error bars represent ± 1 standard deviation, and all sequencing reactions and QS/CRL measurements were performed by GENEWIZ Inc. under blind experimental conditions. QS scores below 24 and CRL scores below 500 indicate problems with sequencing results.

SI Fig. 2

a

b

c

Fig. S2. pBZ22 -/+ Cre – reverse primer sequencing (BZP249)

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

in vivo

in cognito

Induction (min) 120

0 60

120

Contiguous Read Length 0 250 500 750 1000 1250

Pass

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 1,000 Length (bp)


Template

0 10 20 30 40 50

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%reverse%QS%

Quality Score

− Cre

+ Cre

Pass

50 40 30 20 10 0

0 250 500 750 1000 1250

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%reverse%CRL%


− Cre

+ Cre

Pass

250 0 500 750 1000 1250

+ Cre

1 500 1,000 length (bp)

chromatogram alignment

template

− Cre

Fig. S2. pBZ22 -/+ Cre – reverse primer sequencing (BZP249)

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

in vivo

in cognito

Induction (min) 120

0 60

120

Contiguous Read Length 0 250 500 750 1000 1250

Pass

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 1,000 Length (bp)


Template

0 10 20 30 40 50

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%reverse%QS%

Quality Score

− Cre

+ Cre

Pass

50 40 30 20 10 0

0 250 500 750 1000 1250

pBZ22 + pBZ14 - 120 min


pBZ22%DSD2%reverse%CRL%


− Cre

+ Cre

Pass

250 0 500 750 1000 1250

+ Cre

1 500 1,000 length (bp)


template

− Cre

4

Supplementary Figure 3 DNA camouflage with a switchable 2-state device. (a) Quality score (QS) and (b) Contiguous read length (CRL) scores of sequencing reactions of DSD-2[α] maintained in the absence and presence of Crets. (c) The plasmid encoding Crets can be cured out of cells by growing cells at 42°C. All experiments were performed in triplicate, error bars represent ± 1 standard deviation, and all sequencing reactions and QS/CRL measurements were performed by GENEWIZ Inc. under blind experimental conditions. QS scores below 24 and CRL scores below 500 indicate problems with sequencing results.

SI Fig. 3

a

b

c

Fig. #. pBZ22 -/+ pBZ20 (Crets)

3 kb 6 kb

10 kb

DSD-2 Crets

42°C

+--

++-

+-+

+++

DSD-2 Crets

Figures:)Neg62)and)Rxn62)

50 Quality Score

0 10 20 30 40

Pass

− Crets

+ Crets


Pass

0 250 500 750 1,000 1,250

− Crets

+ Crets

– Crets

+ Crets

1 500 1,000 Length (bp)


Template

5

Supplementary Figure 4 DNA camouflage with the 4-state device. (a) In the presence of Cre and Flp, DSD-4[α] was randomly shuffled between α, β, γ, and δ states within a cellular population. (b) Quality score (QS) values of sequencing reactions of DSD-4[α] maintained in the absence and presence of Cre and Flp. (c) Contiguous read length (CRL) scores of sequencing reactions of DSD-4[α] maintained in the absence and presence of Cre and Flp. All experiments were performed in triplicate, error bars represent ± 1 standard deviation, and all sequencing reactions and QS/CRL measurements were performed by GENEWIZ Inc. under blind experimental conditions. QS scores below 24 and CRL scores below 500 indicate problems with sequencing results.

SI Fig. 4

a

b

c

Fig. 2. pBZ23 -/+ pBZ17

in vivo

in cognito

Induction (min)

120 0

60 120


Pass

in vivo

in cognito

Induction (min)

120 0

60 120


Pass

0 250 500 750 1,000 1,250

0

60

120


α

α

α

β

β

β

γ

γ

γ

δ

δ

δ

in cognito

Induction (min)

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 864 Length (bp)


Template

0 10 20 30 40 50

pBZ23 + pBZ17 - 120 min


pBZ23&DSD4&QS&

Quality Score

− Cre-FLP

+ Cre-FLP

Pass

50 40 30 20 10 0

0 250 500 750 1000 1250

pBZ23 + pBZ17 - 120 min


pBZ23&DSD4&CRL&


− Cre-FLP

+ Cre-FLP

Pass

250 0 500 750 1000 1250

0%# 50%# 100%#

120#

60#

0#

alpha#

beta#

gamma#

delta#+ Cre-FLP


α β γ δ

Fig. 2. pBZ23 -/+ pBZ17

in vivo

in cognito

Induction (min)

120 0

60 120


Pass

in vivo

in cognito

Induction (min)

120 0

60 120


Pass

0 250 500 750 1,000 1,250

0

60

120


α

α

α

β

β

β

γ

γ

γ

δ

δ

δ

in cognito

Induction (min)

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 864 Length (bp)


Template

0 10 20 30 40 50

pBZ23 + pBZ17 - 120 min


pBZ23&DSD4&QS&

Quality Score

− Cre-FLP

+ Cre-FLP

Pass

50 40 30 20 10 0

0 250 500 750 1000 1250

pBZ23 + pBZ17 - 120 min


pBZ23&DSD4&CRL&


− Cre-FLP

+ Cre-FLP

Pass

250 0 500 750 1000 1250

0%# 50%# 100%#

120#

60#

0#

alpha#

beta#

gamma#

delta#+ Cre-FLP


α β γ δ

6

Supplementary Figure 5 Shuffling of DSD-2[α]p15A leads to data excision. (a) When DSD-2[α] is placed on a multi-copy plasmid containing a p15A origin (DSD-2[α]p15A), data is maintained in the absence of Cre but excised in the presence of Cre. (b) Quality score (QS) and (c) Contiguous read length (CRL) scores for sequence reactions shown in a. All experiments were performed in triplicate, error bars represent ± 1 standard deviation, and all sequencing reactions and QS/CRL measurements were performed by GENEWIZ Inc. under blind experimental conditions. QS scores below 24 and CRL scores below 500 indicate problems with sequencing results.

SI Fig. 5

a

b

c

Fig. S. pBZ19 -/+ pBZ14

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

0 250 500 750 1,000 1,250

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

Samples:)c,)d,)h,)and)j)

+ Cre

1 500 1,000 length (bp)


template

0 250 500 750 1000 1250

pBZ19 + pBZ14 - 120 min


pBZ19&CRL&


− Cre

+ Cre

Pass

250 0 500 750 1000 1250

0 10 20 30 40 50

pBZ19 + pBZ14 - 120 min


pBZ19&QS&

Quality Score

− Cre

+ Cre

Pass

50 40 30 20 10 0

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 1,000 Length (bp)


Template − Cre

Fig. S. pBZ19 -/+ pBZ14

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

0 250 500 750 1,000 1,250

in vivo

in cognito

Induction (min) 120

0 60

120


Pass

Samples:)c,)d,)h,)and)j)

+ Cre

1 500 1,000 length (bp)


template

0 250 500 750 1000 1250

pBZ19 + pBZ14 - 120 min


pBZ19&CRL&


− Cre

+ Cre

Pass

250 0 500 750 1000 1250

0 10 20 30 40 50

pBZ19 + pBZ14 - 120 min


pBZ19&QS&

Quality Score

− Cre

+ Cre

Pass

50 40 30 20 10 0

in vivo

in cognito

Induction (min)

120

0

60

120

1 500 1,000 Length (bp)


Template − Cre

7

Supplementary Figure 6 Next-generation sequencing (NGS) of 2-state and 4-state devices. (a) Samples 1 and 3: DSD-2[α/β] and DSD4-[α/β/γ/δ] were each separately prepared, purified, and mixed at equal concentration in dH2O. Sample 2 and 4: DSD-2[α] and DSD-4[α] were shuffled with Cre and Cre-Flp recombinases respectively, and then purified, and stored in in dH2O. (b) Samples from (a) run on an agarose gel to demonstrate the purity.

SI Fig. 6

a

b

Fig. 4. Schematic for NGS experiment

Sample 1 2 3 4

DSD-2[α] (F’/oriV,CamR)

DSD-2[β] (F’/oriV,CamR)


+ Cre

(pMB1,KanR)


DSD-4[β] (F’/oriV,CamR)

DSD-4[γ] (F’/oriV,CamR)

DSD-4[δ] (F’/oriV,CamR)


+ Cre-Flp

(pMB1,KanR)

Purify Express Purify Express

Mix Purify Mix Purify

NGS

a

b

Fig. 4. NGS gel

3 kb

1 kb

10 kb

0.5 kb

Sample 1 2 3 4

8

Supplementary Figure 7 NGS identified sequences for Sample 1. Sequences identified by the outside party for Sample 1 (Supplementary Table 4) aligned against (a) DSD-2[α] and (b) DSD-2[β] templates. Gray bars represent areas of perfect sequence alignment and red bars represent areas of sequence misalignment.

SI Fig. 7

a

b

Fig. 4. NGS alignments – Samples 1

DSD-2α DSD-2β

Data Data

CreDSD-2[α]

DSD-2α DSD-2β

Data Data

Cre DSD-2[β]

DSD2-α sequence 1 sequence 2 sequence 3 sequence 4

DSD2-α sequence 1 sequence 2 sequence 3 sequence 4

9

Supplementary Figure 8 NGS identified sequences for Sample 3. Sequences identified by the outside party for Sample 3 (Supplementary Table 4) aligned against (a) DSD-4[α], (b) DSD-4[β], (c) DSD4-[γ], and (d) DSD4-[δ] templates. Gray bars represent areas of perfect sequence alignment and red bars represent areas of sequence misalignment.

SI Fig. 8

a

b

c

d

Fig. 4. NGS alignments – Samples 3

DSD-4α DSD-4β

DSD-4δ DSD-4γ

Data1 Data2 Data3

Data1 Data2 Data3

Data1Data2 Data3

Data1Data2 Data3

FlpCre

DSD-4[α]

DSD-4α DSD-4β

DSD-4δ DSD-4γ

Data1 Data2 Data3

Data1 Data2 Data3

Data1Data2 Data3

Data1Data2 Data3

FlpCre

DSD-4[β]

DSD-4α DSD-4β

DSD-4δ DSD-4γ

Data1 Data2 Data3

Data1 Data2 Data3

Data1Data2 Data3

Data1Data2 Data3

FlpCre DSD-4[γ]

DSD-4α DSD-4β

DSD-4δ DSD-4γ

Data1 Data2 Data3

Data1 Data2 Data3

Data1Data2 Data3

Data1Data2 Data3

FlpCreDSD-4[δ]

DSD4-α

sequence 2 sequence 1

DSD4-β


DSD4-γ


DSD4-δ


10

Supplementary Figure 9 Schematic of the addiction module.

SI Fig. 9

SpyTag'Bla*ssYcbK'SpyCatcher*

Full*complex*

cytoplasm

periplasm

SpyTag'Bla*

ssYcbK'SpyCatcher*

Full*complex*

SpyTag'Bla*

ssYcbK'SpyCatcher*

Full*complex*

covalent assembly and

secretion

constitutive expression

SpyTag

pBR322origin

pBR322origin

Bla YcbK

SpyCatche

r

pBZ51 pBZ52

11

Supplementary Figure 10 pBZ51 and pBZ52 are stably maintained in E. coli. Cells transformed with pBZ51 (selected on Kan), pBZ52 (selected on Kan), and pBZ51 + pBZ52 (selected on Amp) were grown overnight, and plasmid DNA was extracted and run on a 1% agarose gel. Cells co-transformed with pBZ51 and pBZ52 were able to stably maintain both plasmids under Amp selection.

SI Fig. 10

SI Fig. # addiction module plasmids on 1% gel (1hr)

pBZ51 pBZ52

+ − + −

+ +

2 kb 3 kb 4 kb 6 kb

10 kb

pBZ51 pBZ52

12

Supplementary Table 1 NGS analysis of samples 1-4. Over 2 million ~300 bp reads were produced from NGS sequencing of samples 1-4 (Supplementary Fig. 6), with GC contents similar to expected values. DSD-2[α/β]: 9,549 bp/47.8% GC, Cre: 4,452 bp/49.8% GC, DSD4-[α/β/γ/δ]: 8,204 bp/46.8% GC, Cre-Flp: 5,769 bp/46.9% GC.

SI Table 1

SI Table 1

Sample 1 2 3 4

Total Sequences 2,035,696 2,827,422 3,762,818 2,665,635

% GC 48 49 47 46

13

Supplementary Table 2 Assembly of NGS reads from samples 1-4. Here, the statistics of the assembled scaffolds from are shown.

SI Table 2

Table 1

Sample 1 2 3 4

Sequence size 4,484,782 109,143 4,575,261 238,314

Number of scaffolds 711 248 500 536

% GC 50.7 49.3 50.7 50.1

Shortest contig size 301 300 306 300

Median sequence size 3,897 360 3,943 390

Mean sequence size 6,307.7 440.1 9,150.5 444.6

Longest contig size 51,023 5,385 93,737 5,397

Number of subsystems 564 2 576 2

Number of coding sequences 4,300 64 4,410 190

Number of RNAs 34 0 30 0

14

Supplementary Table 3 Identification of annotated and assembled samples 1-4. Since there was no prior information provided regarding samples 1-4, the assembled scaffolds (Supplementary Table 2) were blasted against a plasmid database (http://plasmid.med.harvard.edu/) by the outside party. Identified hits were based on >90% sequence identity and a minimum of 100 bp alignment length.

SI Table 3 SI Table 2

Sample Total Scaffolds

Aligned Scaffolds

% Aligned

Identified Vectors

1 711 12 1.7 •  pBluescriptR (AmpR) •  pDONR221 (KanR) •  pOTB7 (CamR)



4 536 6 1.1 •  pBluescriptR (AmpR) •  pDONR221 (KanR) •  pOTB7 (CamR) •  pK7-GFP (AmpR)

15

Supplementary Table 4 Identified sequences by the outside party following NGS analysis and sequence assembly. These sequences were assembled once the sequence of the backbone vectors were provided to the outside party.

SI Table 4 Fig. NGS Sample 1-4 identified sequences

Sample Sequence Number Identified Insert

1

1

TTCATCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATGGTATATCTCCTTCTTAAAGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTAAATTG

2 ATAACTTCGTATAATGTATGCTATACGAAGTTATGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACAATTGTGAGCGGATAACAATTTACATTGTGAGCGGATAACAAGATACTGAGCACATCAGCAGGACGCACTGACC

3 ATAACTTCGTATAATGTATGCTATACGAAGTTATGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAGGTATCTGGCACTACGTTCAGGTAACCTGAAGCTCGAATCCAGTACTCGACGTC

4 ATAACTTCGTATAATGTATGCTATACGAAGTTATGCTAGCTATGGAGAAACAGTAGAGAGTTGCGATAAAAAGCGTCAGGTAGGATCCGCTAATCTTATGGATAAAAATGCTATGGCATAGCAAAGTGTGACGCCGTGCAAATAATCAATGTGGACTTTTCTGCCGTGATTATAGACACTTTTGTTACGCGTTTTTGTCATGGCTTTGGTCCCGCTTTGTTACAGAATGCTTTTAATAAGCGGGGTTACCGGTTTGGTTAGCGAGAAGAGCCAGTAAAAGACGCAGTGACGGCAATGTCTGATGCAATATGGACAATTGGTTTCTTCGTCGGTCTGATTATTAGTCTGGG

2 no insert sequence identified

3

1

TTCATCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATGGTATATCTCCTTCTTAAAGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTAAATTGTTATCCGCTCACAATTGTATCCGCTCATGAATTAATTCTTAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCAGGCATTGATGGAATCGTAGTCTCAATAACTTCGTATAGCATACATTATACGAAGTTAT

2

TTCATCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATGGTATATCTCCTTCTTAAAGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTAAATTGTTATCCGCTCACAATTGTATCCGCTCATGAATTAATTCTTAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCCTCATCGAGCATCAAATGAAACTGCATAACTTCGTATAGCATACATTATACGAAGTTAT

4 no insert sequence identified

16

Construct Plasmid Name

Plasmid Backbone

Sequence Legend

Cre pBZ14 pET28a (pBR322 origin and KanR only)

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCAGCAGGACGCACTGACCACTTTAAGAAGGAGATATACCATGGCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAAGTCGACAACCTAGGAAAAACCTGAGGAAAATGCATAGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGCTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAA

PLtetO-1 Cre Terminator Spacer

Crets pBZ20 pKD46 (origin and

AmpR only)

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCAGCAGGACGCACTGACCACTTTAAGAAGGAGATATACCATGGCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAAGTCGACAACCTAGGAAAAACCTGAGGAAAATGCATAGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGCTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAA

PLtetO-1 Cre Terminator Spacer

Cre-Flp pBZ17 pET28a (pBR322 origin and KanR only)

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCAGCAGGACGCACTGACCACTTTAAGAAGGAGATATACCATGGCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAATAAAAGCTTCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGAGCCAATTTGATATATTATGTAAAACACCACCTAAGGTCCTGGTTCGTCAGTTTGTGGAAAGGTTTGAAAGACCTTCAGGGGAAAAAATAGCATCATGTGCTGCTGAACTAACCTATTTATGTTGGATGATTACTCATAACGGAACAGCAATCAAGAGAGCCACATTCATGAGCTATAATACTATCATAAGCAATTCGCTGAGTTTCGATATTGTCAACAAATCACTCCAGTTTAAATACAAGACGCAAAAAGCAACAATTCTGGAAGCCTCATTAAAGAAATTAATTCCTGCTTGGGAATTTACAATTATTCCTTACAATGGACAAAAACATCAATCTGATATCACTGATATTGTAAGTAGTTTGCAATTACAGTTCGAATCATCGGAAG

PLtetO-1 Cre Flp Terminator Spacer

17

AAGCAGATAAGGGAAATAGCCACAGTAAAAAAATGCTTAAAGCACTTCTAAGTGAGGGTGAAAGCATCTGGGAGATCACTGAGAAAATACTAAATTCGTTTGAGTATACCTCGAGATTTACAAAAACAAAAACTTTATACCAATTCCTCTTCCTAGCTACTTTCATCAATTGTGGAAGATTCAGCGATATTAAGAACGTTGATCCGAAATCATTTAAATTAGTCCAAAATAAGTATCTGGGAGTAATAATCCAGTGTTTAGTGACAGAGACAAAGACAAGCGTTAGTAGGCACATATACTTCTTTAGCGCAAGGGGTAGGATCGATCCACTTGTATATTTGGATGAATTTTTGAGGAACTCTGAACCAGTCCTAAAACGAGTAAATAGGACCGGCAATTCTTCAAGCAACAAACAGGAATACCAATTATTAAAAGATAACTTAGTCAGATCGTACAACAAGGCTTTGAAGAAAAATGCGCCTTATCCAATCTTTGCTATAAAGAATGGCCCAAAATCTCACATTGGAAGACATTTGATGACCTCATTTCTGTCAATGAAGGGCCTAACGGAGTTGACTAATGTTGTGGGAAATTGGAGCGATAAGCGTGCTTCTGCCGTGGCCAGGACAACGTATACTCATCAGATAACAGCAATACCTGATCACTACTTCGCACTAGTTTCTCGGTACTATGCATATGATCCAATATCAAAGGAAATGATAGCATTGAAGGATGAGACTAATCCAATTGAGGAGTGGCAGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGAAGCATACGATACCCCGCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCATCCTACATAAATTAATAAGTCGACAACCTAGGAAAAACCTGAGGAAAATGCATAGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGCTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAA

DSD-2[α] pBZ22 pBAC-LacZ

(F’/oriV origins and

CamR)

ATAACTTCGTATAATGTATGCTATACGAAGTTATGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAGGTATCTGGCACTACGTTCAGGTAACCTGAAGCTCGAATCCAGTACTCGACGTCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTCTAGCACGCGTACCTGGTGGCGCGCCTTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATGGTATATCTCCTTCTTAAAGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTAAATTGTTATCCGCTCACAATTGTATCCGCTCATGAATTAATTCTTAGGCATATTCAAATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATAAACGCTACACAGGCTCCTGAGATTAATAATGCGGATCTGTCCCAGACTAATAATCAGACCGACGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATAGCTAGCATAACTTCGTATAGCATACATTATACGAAGTTAT

loxP Data

DSD-2[α]p15A pBZ19 p15A (origin and

CamR only)

ATAACTTCGTATAATGTATGCTATACGAAGTTATGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAGGTATCTGGCACTACGTTCAGGTAACCTGAAGCTCGAATCCAGTACTCGACGTCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTCTAGCACGCGTACCTGGTGGCGCGCCTTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATGGTATATCTCCTTCTTAAAGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTAAATTGTTATCCGCTCACAATTGTATCCGCTCATGAATTAATTCTTAGGCATATTCAAATCGTTTTCGTTACCGCTTGCAGGCATCATGACAGAACACTACTTCCTATAAACGCTACACAGGCTCCTGAGATTAATAATGCGGATCTGTCCCAGACTAATAATCAGACCGACGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATAGCTAGCATAACTTCGTATAGCATACATTATACGAAGTTAT

loxP Data

DSD-4[α] pBZ23 pBAC-LacZ

(F’/oriV

ATAACTTCGTATAATGTATGCTATACGAAGTTATGCAGTTTCATTTGATGCTCGATGAGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAAGCTCGAATCCAGTACTCGACGTCTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTCTAGCACGCGTACCTGGTGGCGCGCCTTATTTGTATAGTTC

loxP FRT Data1

18

origins and CamR)

ATCCATGCCATGTGTAATCCCAGCAGCTGTTACAAACTCAAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGATTGTGTGGACAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTTGTTGATAATGGTCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTAACTTTGATTCCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTATTCCAATTTGTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTCTATTAACAAGGGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCTTTGAAAAATATAGTTCTTTCCTGTACATAACCTTCGGGCATGGCACTCTTGAAAAAGTCATGCTGTTTCATATGATCTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGTTGGCCATGGAACAGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTTGCATCACCTTCACCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAACAAGAATTGGGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATGGTATATCTCCTTCTTAAAGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCTTGTTATCCGCTCACAATGTAAATTGTTATCCGCTCACAATTGTATCCGCTCATGAATTAATTCTTAGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCAGGCATTGATGGAATCGTAGTCTCAATAACTTCGTATAGCATACATTATACGAAGTTAT

Data2 Data3

SpyTag-Bla pBZ51 pET28a (pBR322 origin and KanR only)

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCAGCAGGACGCACTGACCACTTTAAGAAGGAGATATACCATGGCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAGGGTTCAGGGGGTTCCGGTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTGATAAGTCGACAACCTAGGAAAAACCTGAGGAAAATGCATAGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGCTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAA

PLtetO-1 SpyTag Linker Bla Terminator

YcbK-SpyCatcher

pBZ52 pET28a (pBR322 origin and KanR only)

TCCCTATCAGTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCAGCAGGACGCACTGACCACTTTAAGAAGGAGATATACCATGGATAAATTTGATGCGAACCGCCGCAAACTGCTGGCGCTGGGCGGCGTGGCGCTGGGCGCGGCGATTCTGCCGACCCCGGCGTTTGCGACCCTGAGCACCCCGCGCGGAAGCGGTAGTGGAAGTATGGGAGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACGCTCATATTTAATGATAAGTCGACAACCTAGGAAAAACCTGAGGAAAATGCATAGCTAGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGCTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAA

PLtetO-1 YcbK SpyCatcher Terminator

MIT Message 1


GACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCACCTCGAGCTGGTGGCGCGCCTTATTTGTATAGTGGCCACGATCCATGCTAACGTCTCTGCGTAGGGATGAATCCCGTTTTGAACTCGTTCCTACTGACGGACGAGCTGATAGGTAGCCGAAGTAGTGATACGATCCACACATGCCATCATTGCATACTCGTGCATTCAATGATGCATAGTCACGTAGTCCATATGGTAATGGTGATGTCAAGTCACATGTCAATACTCGTCACTAGAACTGAGCGCGATGACTGGCGAGCTGGTGCGCTCCCGAGGCTGGTCGAGCGACTAAGTTGAATGCGCAGACCGATCGAGACGACTCTAGCGCTGGAATAAATCAGAATAAAGACCTAGGGATATATTCCGCTTCGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGG

Forward primer MIT message 1 Reverse primer

MIT Message 2


GACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCACCTCGAGCTGGTGGCGCGCCTTATTTGTATAGCCCACCAATACTGCCAATAGACGGTACTGTACACCCTGTTTTACAGCAACGGGAAAGGAGGATCACTTTCTACAATTGTGTGCTGGACTGACAGTCGCATATCCACACATGCCATCATTGCATACTCGTGCATTCAATGATGCATCTACACGTAGTCCATATGGTAATGGTGATGTCACTACACATGTCAATACTCGTCACTAGAACTGAGCGCGATACGACTCGCCCATAGGGTTCGCCGGCTCGCACTGACTACCTTACGCTCTGACCCAGATCGGAGCCGGCCGCATGACCCCTGTGATATAATACCGTTCATCCCTAGGGATATATTCCGCTTCGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGG

Forward primer MIT message 2 Reverse primer

Supplementary Table 5 Identity, plasmid, and sequence information of all constructs used in this study.

DNA Camouflage - DTIC · all sequencing reactions and QS/CRL measurements were performed by GENEWIZ...

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