CONTENT PAGE
PAGE NO.
Predicting the Effect of RBS on Transfer Function of Input Devices...……………. 3
Supplementary Table 1. Transfer functions of input switch devices derived from
experimental results and model prediction….......................................................... 4
Supplementary Fig. 1. Predicting the transfer functions of input devices with different
ribosome binding sites…………………………………………………………….
5
Supplementary Fig. 2. Design of input device with strong and weak ribosome binding
sites……………………………………………………………………………........
6
Supplementary Fig. 3. Design of input device B with strong and weak ribosome
binding sites………………………………………………………………………...
7
Supplementary Fig. 4. Pairing compatibility assessment of (A) pRHAB promoter with
RFP expression, and (B) pBAD promoter with GFP expression…………......
8
Supplementary Fig. 5. Effects of genetic architecture on the switching behaviour of
σ54-dependent pHrpL promoter....................................................................
9 – 10
Supplementary Fig. 6. Directed evolution and characterisation of lambda repressor
binding sites...................................................................................................
11
Supplementary Fig. 7. Flow cytometry analysis of the biological half adder on a 2D
contour plot....................................................................................................
12
Supplementary Fig. 8. The effect of plasmid copy number in the shunting and
sequestering of transcription factors..............................................................
13
Supplementary Fig. 9. Cell viability profile of the biological half adder after 4 hours of
induction.........................................................................................................
14
1
Predicting the Effect of RBS on Transfer Function of IMPLY Gates...............
15
Supplementary Table 2. List of parameters used in the modelling of IMPLY logic
gates................................................................................................................
15
Supplementary Fig. 10: Predicted transfer functions of the IMPLY gate with different
ribosome binding sites at steady state.............................................................
16
Model-aided Design of AND, OR & XOR Logic Gates................................... 17 – 22
Supplementary Table 3. Transfer functions of input switch devices in the modelling of
AND and OR logic gate..................................................................................
18
Supplementary Fig. 11. Predicted normalised output of HrpRS AND gate.....
19
Supplementary Fig. 12. Predicted normalised output of pBAD-pRHAB OR
gate.................................................................................................................
21
Supplementary Fig. 13. Predicted normalised outputs of 4 different OR gate
designs……………………………………………………………………………....
22
Sequence Design……………………………………………………………………….... 24
References.............................................................................................................. 47
2
Predicting the Effect of RBS on Transfer Function of Input Devices
To enable model-driven design and optimization of synthetic biological systems with
more complicated circuitry and computational functions, we examine the effect of
ribosome binding sites (RBS) on the steady state transfer function of input switch
devices. By analysing reference data [1] that had previously characterise the inputoutput relationship of genetic switches in the form
Output Y = A +
B(X n )
Cn + X n
𝐄𝐪𝐧. (1)
Where A, B, C and n are empirically derived parameter and X is input concentration,
, we observed that parameters that are most sensitive to changes in RBS are
parameters A and B. Accordingly, knowing the relative output of switch devices with
weaker RBS by either prediction from reliable software or by single experimental
measurement of device’s output at input maximal, the parameters A and B can be
scaled proportionally to obtain a priori parameters that accurately describe the transfer
function of other devices with weaker RBS (Supplementary Fig. 1A). We validated our
approach with previously obtained data sets (Supplementary Table 1) and showed
that the transfer function of input devices pLuxR (Supplementary Fig. 1B) and pBAD
(Supplementary Fig. 1C) with different RBS can be reliably estimated without
excessive experimentation.
3
Supplementary Table 1. Transfer functions of input switch devices derived from
experimental results and model prediction. Input devices marked with * were used to
derive the values of parameter A and B for other devices with the same inducible
promoter but with different RBS.
Input Device
A
B
C (mM)
n
R2
Ref
pLuxR-Rbs34-GFP*
2010.01
1.349E+05
2.890E-09
1.898
0.994
[1]
pLuxR-Rbs31-GFP
969.32
7.693E+04
2.955E-09
1.771
0.999
[1]
pLuxR-Rbs32-GFP
581.16
5.143E+04
3.509E-09
1.655
0.999
[1]
pLuxR-RbsH-GFP
364.12
3.194E+04
3.784E-09
1.616
0.998
[1]
pLuxR-Rbs34-GFP
2010.01
1.349E+05
2.890E-09
1.898
0.994
This work
pLuxR-Rbs31-GFP
1198.15
7.693E+04
2.890E-09
1.898
0.986
This work
pLuxR-Rbs32-GFP
801.00
5.143E+04
2.890E-09
1.898
0.965
This work
pLuxR-RbsH-GFP
497.45
3.194E+04
2.890E-09
1.898
0.946
This work
pBAD-Rbs34-GFP*
705.50
1.411E+05
5.240E-04
1.173
0.999
[1]
pBAD-RbsH-GFP
304.56
9.229E+05
4.800E-04
1.415
0.999
[1]
pBAD-Rbs32-GFP
135.41
5.208E+04
5.160E-04
1.268
0.999
[1]
pBAD-Rbs33-GFP
16.77
1.290E+04
5.130E-04
1.323
0.999
[1]
pBAD-Rbs34-GFP
705.50
1.411E+05
5.240E-04
1.173
0.999
This work
pBAD-RbsH-GFP
461.45
9.229E+05
5.240E-04
1.173
0.947
This work
pBAD-Rbs32-GFP
260.40
5.208E+04
5.240E-04
1.173
0.979
This work
pBAD-Rbs33-GFP
64.50
1.290E+04
5.240E-04
1.173
0.983
This work
4
A
GFP/Cell (au)
1.6E+05
1.4E+05
1.2E+05
1.0E+05
B
GFP/Cell (au)
1.6E+05
Empirical Fit pLuxR-Rbs34-GFP
Empirical Fit pluxR-Rbs31-GFP
Empirical Fit pLuxR-Rbs32-GFP
Empirical Fit pLuxR-RbsH-GFP
Model Fit pLuxR-Rbs34-GFP
Model Fit pLuxR-Rbs31-GFP
Model Fit pluxR-Rbs32-GFP
R2 = 0.994
1.4E+05
1.2E+05
1.0E+05
R2 = 0.986
8.0E+04
R2 = 0.965
6.0E+04
4.0E+04
R2 = 0.946
4.0E+04
0.0E+00
1.0E-13
1.0E-09
3OC6HSL (M)
1.0E-07
1.0E-05
R2 = 0.947
R2 = 0.979
R2 = 0.983
2.0E+04
1.0E-11
R2 = 0.999
8.0E+04
6.0E+04
2.0E+04
Empirical Fit pBAD-Rbs34-GFP
Empirical Fit pBAD-RbsH-GFP
Empirical Fit pBAD-Rbs32-GFP
Empirical Fit pBAD-Rbs33-GFP
Model Fit pBAD-Rbs34-GFP
Model Fit pBAD-RbsH-GFP
Model Fit pBAD-Rbs32-GFP
Model Fit pBAD-Rbs33-GFP
0.0E+00
1.0E-08 1.0E-07 1.0E-06 1.0E-05 1.0E-04 1.0E-03 1.0E-02 1.0E-01
Arabinose (M)
Supplementary Fig. 1. Predicting the transfer functions of input devices with different ribosome binding sites (RBS). Validation of the suggested
forward engineering approach with (A) pLuxR and (B) pBAD expression systems with different ribosome bind sites upstream of GFP reporter.
Solid lines represent the forward predicted transfer functions, with corresponding R-squared values, while symbols represent empirically-derived
transfer functions using total experimental fitting. All the empirically-derived transfer functions have R-squared values of 0.998 or greater and are
obtained from an earlier study by Wang et al 2011.
5
A
High Copy
pCON
RhaS
AraC
RbsA
pBAD
RFP
High Copy
B
pCON
RhaS
AraC
RbsB
pBAD
RFP
C
RFP/Cell (au)
4900
4200
Experimental pBAD-RbsA-RFP
Transfer Function pBAD-RbsA-RFP
Transfer Function pBAD-RbsB-RFP
3500
RbsA > RbsB
R2 = 0.999
2800
2100
1400
700
0
1.00E-07
1.00E-06
1.00E-05
1.00E-04
1.00E-03
Arabinose (M)
1.00E-02
1.00E-01
Supplementary Fig. 2. Design of input device A with (A) strong and (B) weak
ribosome binding sites, respectively. Input device A is modelled after the arabinoseinduced expression system. In the presence of arabinose, AraC binds and triggers
conformational change in the pBAD promoter to activate RFP expression. The RhaS
transcription factor (for use with rhamnose-inducible expression system) is included in
the design to enable accurate characterisation of input device B in the context of the
overall half adder genetic circuit. (C) Transfer function of the pBAD promoter. Green
triangles represent experimental data, while black line represents empirically-derived
transfer function for construct with a strong ribosome binding site - as denoted in the
equation above. The blue line represents the predicted transfer function for a construct
with a weak ribosome binding site. Error bars represent the standard deviation of
biological triplicates.
6
A
High Copy
RbsA
pCON
RhaS
AraC
RFP
pRHAB
High Copy
B
RbsB
pCON
RhaS
AraC
RFP
pRHAB
C
RFP/Cell (au)
7000
6000
5000
Experimental pRHAB-RbsA-RFP
Transfer Function pRHAB-RbsA-RFP
Transfer Function pRHAB-RbsB-RFPB
RbsA > RbsB
R2 = 0.998
4000
3000
2000
1000
0
1.00E-07
1.00E-06
1.00E-05
1.00E-04
1.00E-03
Rhamnose (M)
1.00E-02
1.00E-01
Supplementary Fig. 3. Design of input device B with (A) strong and (B) weak
ribosome binding sites, respectively. Input device B is modelled after the rhamnoseinduced expression system. In the presence of rhamnose, RhaS binds and triggers
conformational change in the pRHAB promoter to activate RFP expression. The AraC
transcription factor (for use with arabinose-inducible expression system) is included in
the design to enable accurate characterisation of input device B, in the context of the
overall half adder genetic circuit. (C) Transfer function of pRHAB promoter. Blue
diamonds represent experimental data, while black line represents an empiricallyderived transfer function for a construct with strong ribosome binding site, as denoted
in the equation above. The red line represents the predicted transfer function for
construct with weak ribosome site. Error bars represent the standard deviation of
biological triplicates.
7
High Copy
pCON
RhaS
AraC
RbsA
pRHAB
RbsA
RFP
pBAD
GFP
A
RFP/Cell (au)
7200
6000
Variable Rhamnose and 0.02% Arabinose
Variable Rhamnose
Variable Arabinose
4800
3600
2400
1200
0
1.00E-07
1.00E-06
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E-03
1.00E-02
1.00E-01
Inducer (M)
B
GFP/Cell (au)
24000
20000
Variable Arabinose and 0.02% Rhamnose
Variable Arabinose
Variable Rhamnose
16000
12000
8000
4000
0
1.00E-07
1.00E-06
1.00E-05
1.00E-04
Inducer (M)
Supplementary Fig. 4. Pairing compatibility assessment of (A) pRHAB promoter with
RFP expression, and (B) pBAD promoter with GFP expression. The red squares and
green triangles show the characteristics of each promoter following induction with its
natural and heterologous sugar, respectively. The blue diamonds represent the
performance of each input device in the presence of both sugars. Error bars represent
standard deviation of biological triplicates.
8
GENETIC CONSTRUCT
A
pHrpL
GFP
OUTPUT
OFF
OFF
High Copy
B
GFP
pHrpL
λCl
OFF
OFF
pHrpL
High Copy
C
λCl
pHrpL
GFP
pHrpL
ON
ON
High Copy
D
λCl
GFP
OFF
OFF
pHrpL
High Copy
E
GFP
λCl
pHrpL
pHrpL
OFF
High Copy
Low Copy
GFP
GFP
F
pHrpL
High Copy
pHrpL
OFF
OFF
Low Copy
9
G
GFP/Cell (au)
300
250
200
150
100
50
0
pHrpL-GFP (HC)
pHrpL-GFP-pHrpL-λcl
(HC)
pHrpL-λcl-pHrpL-GFP
(HC)
λcl-pHrpL-GFP (HC)
pHrpL-GFP (LC)
pHrpL-λcl (HC)
pHrpL-GFP (LC & HC)
Supplementary Fig. 5. Effects of genetic architecture on the switching behaviour of σ54-dependent pHrpL promoter. (A) pHrpL-GFP reporter in
high copy plasmid, (B) pHrpL-GFP reporter assembled upstream of pHrpL-λCl in high copy plasmid, (C) pHrpL-GFP reporter assembled
downstream of pHrpL-λCl in high copy plasmid, (D) pHrpL-GFP reporter assembled downstream of λCl in high copy plasmid, (E) pHrpL-GFP
reporter and pHrpL-λCl separately assembled in low and high copy plasmids, respectively, and (F) pHrpL-GFP reporter assembled in both low and
high copy plasmids. Within the same plasmid vector, an pHrpL promoter placed upstream is able to turn on a proximate, downstream pHrpL
promoter and caused GFP expression - even in the absence of HrpRS transcription activators. This genetic context dependent effect can be
circumvented by insulating pHrpL expression cassettes in different plasmids. (G) Quantitative characterisation of pHrpL promoter leakiness in
different genetic contexts. Error bars represent the standard deviation of three independent experiments.
10
A
B
Low Copy
RFP/Cell (au)
350
HrpS
pBAD
HrpR
pRHAB
pCON
RhaS
pBADcl2A-RFPasv
pBADcl2B-RFPasv
AraC
300
High Copy
250
+1
pBAD .. ATATATCACCGGCGGTGATA
200
λCl2A
pHrpL
λCl
RbsB RFPASV
pBAD
150
Directed
Evolution
100
+1
pBAD .. ATATATCACCGGGCGTGCTG
50
λCl2B
pHrpL
λCl
pBAD
RbsB RFPASV
0
NC
A
R
A+R
(NO INPUT)
(INPUT A)
(INPUT B)
(INPUT A & B)
Supplementary Fig. 6. Directed evolution and characterisation of lambda repressor binding sites. (A) Layout of genetic circuit used in the mutation
and screening of lambda repressor binding sites. Characteristic DNA sequences of the lambda repressor binding sites with perfect dyad symmetry
were mutated and screened for RFP expression in the presence of arabinose (input A) and suppression of RFP in the presence of both arabinose
and rhamnose (input B). Lambda repressor binding sites located downstream of pBAD promoter transcription start site (denoted with +1) are
denoted as red and green letters (reverse complement). Key mutations that generated the best repressor in this screen are marked by green boxes.
(B) Characterisation of original (blue bars) and evolved lambda repressor binding sites (red bars) in the engineered genetic circuit. Error bars
represent the standard deviation of biological triplicate.
11
A
B
105
FITC-A
104
104
102
103
102
PE-CF594-A
PE-CF594-A
103
105
FITC-A
102
103
104
105
102
C
103
104
105
104
105
D
102
103
102
103
104
105
FITC-A
PE-CF594-A
PE-CF594-A
104
105
FITC-A
102
103
104
105
102
103
Supplementary Fig. 7. Flow cytometry analysis of the biological half adder on a 2D
contour plot. (A) Half adder without any inputs, (B) half adder with only arabinose (input
A), (C) half adder with only rhamnose (input B) and (D) half adder with both inputs A
and B. FITC-A and PE-CF594-A represent channels that detect GFP and RFP
fluorescence, respectively.
12
GFP/Cell (au)
1000
Half Adder I
AND Gate
800
600
400
200
0
NC
(NO INPUT)
A
(INPUT A)
R
(INPUT B)
A+R
(INPUT A & B)
Supplementary Fig. 8. The effect of plasmid copy number in the shunting and
sequestering of transcription factors. GFP output of the pure AND gate is higher than
the GFP output of half adder’s AND gate. Error bars represent the standard deviations
for four independent experiments.
13
Viable Cells
(cful/ml)
4.0E+09
3.0E+09
2.0E+09
1.0E+09
0.0E+00
NC
(NO INPUT)
A
(INPUT A)
R
(INPUT B)
A+R
(INPUT A & B)
Supplementary Fig. 9. Cell viability profile of the biological half adder after 4 hours of
induction. Error bars represent the standard deviations for four independent
experiments.
14
Predicting the Effect of RBS on Transfer Function of NIMPLY Gates
We characterised the steady state transfer function of the NIMPLY gate with strong
RBS by titrating cells that were activated for RFP expression by a fixed input of B
above switch point with variable concentration of input A (Fig. 4C). Our result shows
that the NIMPLY gate can be described mathematically by a Hill-like equation of the
form
IMPLY = A +
αB(Cn )
Cn + X n
IMPLY = 571 +
𝐄𝐪𝐧. (2)
2278α(5.66 × 10−05 )0.718 )
(5.66 × 10−05 )0.718 + [Ara]0.718
𝐄𝐪𝐧. (3)
Where A, B, C and n are empirically derived parameter, X is input A concentration,
and α = 1 in presence and 0 in absence of input B above switch point.
Drawing insights from earlier results, we hypothesized that parameters A and B are
parameters that are most sensitive to the overall behaviour of NIMPLY gates when the
RBS that initiate the expression of the RFP reporter are changed. To test our
hypothesis, we simulated the output of NIMPLY gates whereby the RFP expressions
are initiated from RBS of different strengths (modelling parameters as listed in
Supplementary Table 2). The modelling results (Supplementary Fig. 10) are coherent
with observed experimental results and show that RBS can be used for fine tuning of
layered genetic circuits.
Supplementary Table S2. List of parameters used in the modelling of NIMPLY logic
gates. NIMPLY circuit marked with * was empirically fitted using least square
regression fitting (R2 = 0.998), and used to derive the values of parameter A and B for
other NIMPLY circuits in order of decreasing output.
IMPLY GENETIC CIRCUIT
A
B
C (M)
n
pBAD-Rbs34-λCl-T-pBAD-pRHAB-RbsA1-RFP-T*
571
2278
5.66E-05
0.718
pBAD-Rbs34-λCl-T-pBAD-pRHAB-RbsA2-RFP-T
420
1708
5.66E-05
0.718
pBAD-Rbs34-λCl-T-pBAD-pRHAB-RbsA3-RFP-T
270
1139
5.66E-05
0.718
pBAD-Rbs34-λCl-T-pBAD-pRHAB-RbsB-RFP-T
120
570
5.66E-05
0.718
15
RFP Expression
(RFU/OD)
3.0E+03
RBS Strength: A1, A2, A3, B,
(from strongest to weakest)
pBAD-Lcl-pBADpRHABcl2b-RbsA1-RFP
pBAD-Lcl-pBADpRHABcl2b-RbsA2-RFP
pBAD-Lcl-pBADpRHABcl2b-RbsA3-RFP
pBAD-Lcl-pBADpRHABclb2-RbsB-RFP
2.5E+03
2.0E+03
1.5E+03
1.0E+03
5.0E+02
0.0E+00
1.0E-07
1.0E-06
1.0E-05
1.0E-04
1.0E-03
Arabinose (M)
1.0E-02
1.0E-01
Supplementary Fig. 10: Predicted transfer functions of the NIMPLY gate with different
ribosome binding sites at steady state. Transfer function of the NIMPLY gate with the
strongest ribosome binding site RbsA1 was obtained by empirical fitting.
16
Model-aided Design of AND, OR & XOR Logic Gates
To model an AND logic gate, transfer function of individual switch devices were
incorporated to the following equation similar to how the effective resistance of a
parallel arrangement of resistors was calculated.
ANDAbs = [
αAND
1
1
+
TF1 TF2
]
𝐄𝐪𝐧. (4)
Where αAND is an empirically-derived parameter, and TF1 and TF2 are transfer
functions of input switch device 1 and 2 respectively.
This modelling approach is based on the assumption that at steady state, intracellular
genetic expression occurs on a faster time scale than diffusion of inducers into the
cells. Thus, the key factor which influences the characteristic and performance of
genetic logic gates follows the transfer function of input genetic switches.
From Eqn. (1), the transfer functions of input 1 and 2 are presented as,
TF1 = A1 +
TF2 = A2 +
B1 (X1 n1 )
C1 n1 + X1 n1
B2 (X2 n2 )
C2 n2 + X2 n2
𝐄𝐪𝐧. (5)
𝐄𝐪𝐧. (6)
Substituting, Eqn. (5) and (6) into Eqn. (4), the normalised output of AND logic gate:
ANDNorm =
α
[ 1 AND 1 ]
TF1 + TF2
lim
X1 X2 →∞
α
[ 1 AND 1 ]
TF1 + TF2
17
1
1
1
+
n1
B1 (X1 )
B2 (X2 n2 )
A1 + n1
A
+
2
[
C1 + X1 n1
C2 n2 + X2 n2 ]
=
lim
𝐄𝐪𝐧. (6)
1
X1 X2 →∞
1
1
+
B1 (X1 n1 )
B2 (X2 n2 )
A1 + n1
A2 + n2
[
C1 + X1 n1
C2 + X2 n2 ]
From Eqn. (5) and (6), as X1 and X2 approaches infinity, TF1 and TF2 are B1 and B2
respectively. Thus, the normalised output of AND logic gate is derived as:
ANDNorm = [
B1 + B2
]
1
1
B1 B2 (TF1 + TF2)
𝐄𝐪𝐧. (7)
Supplementary Table 3. Transfer functions of input switch devices in the modelling
of AND and OR logic gates.
Input Device
(Rbs)
A
B
C
n
Used in Modelling
pBAD-(strong RBS)
15
3885
1.16E-4
1.983
OR GATE
pBAD-(weak RBS)
2.8
732
1.16E-4
1.983
AND GATE
pRHAB-(strong RBS)
45
5906
4.31E-4
0.914
OR GATE
pRHAB-(weak RBS)
4.6
783
4.31E-4
0.914
AND GATE
Normalised outputs of a hypothetical AND gate design were modelled with transfer
functions from 2 input switch devices of varying expression strength as listed in
Supplementary Table 3. Supplementary Fig. 11 shows the predicted output of a
hypothetical HrpRS AND gate. The model agreed well with experimental results (Fig.
2B) with a R2 value of 0.94.
18
Supplementary Fig. 11. Predicted normalised output of HrpRS AND gate. The model
was validated with a R2 value of 0.94.
19
To model an OR logic gate, transfer function of individual switch devices were
incorporated to the following equation similar to how the effective resistance of serial
arrangement of resistors was calculated.
OR Abs = [αOR (TF1 + TF2)]
𝐄𝐪𝐧. (8)
Substituting, Eqn. (5) and (6) into Eqn. (8), the normalised output of OR logic gate:
OR Norm =
[αOR (TF1 + TF2)]
lim [αOR (TF1 + TF2)]
X1 X2 →∞
(TF1 + TF2)
=
lim
X1 X2 →∞
B1 (X1 n1 )
B2 (X2 n2 )
[A1 + n1
+ A2 + n2
]
C1 + X1 n1
C2 + X2 n2
𝐄𝐪𝐧. (9)
Thus, the normalised output of OR logic gate is derived as:
(TF1 + TF2)
OR Norm = [
]
B1 + B2
𝐄𝐪𝐧. (10)
Normalised outputs of a hypothetical OR gate were modelled with transfer functions
from 2 input switch devices of strong expression strength as listed in Supplementary
Table 3. Fig. 12 shows the predicted output of a hypothetical pBAD-pRHAB OR gate.
The model agreed well with experimental results (Fig. 3B) with a R2 value of 0.96.
20
Supplementary Fig. 12. Predicted normalised output of pBAD-pRHAB OR gate. The
model was validated with a R2 value of 0.96.
21
Supplementary Fig. 13. Predicted normalised outputs of 4 different OR gate designs
with transfer functions corresponding to (A) TF1W-TF2S, (B) TF1S-TF2W, (C) TF1WTF2W, and (D) TF1S-TF2S. From the model, only design C and D are functional OR
gates, while design A and B are imbalanced in the overall expression profile.
22
The XOR logic gate was designed by using λCl repressors generated from HrpRS
AND gate to inhibit output from the OR logic gate. Thus, outputs of XOR logic could
be modelled as a difference of OR and AND outputs:
XOR Abs = OR Abs − ANDAbs
𝐄𝐪𝐧. (8)
In the presence of both input A and B, an ideal XOR logic would display an output of
zero. Thus, in order for layered logic gates to perform XOR logic operation when both
input A and B are introduced,
OR Abs − ANDAbs ≤ 0
OR Abs ≤ ANDAbs
(condition for XOR)
𝐄𝐪𝐧. (9)
The arabinose promoter had a maximum expression of ~20,000au GFP in the
presence of input B (Supplementary Fig. 4B). Comparatively, pHrpL promoter in a high
copy plasmid exhibited an expression of ~4000au GFP in the presence of both input
A and B (Fig. 2D). By interpolating 4000au GFP expression on the Y-axis of the
characterisation curve of pBAD promoter (Supplementary Fig. 4B), we estimated that
the pHrpL promoter had an expression strength that corresponded to pBAD that was
induced by 5.00E-05M arabinose on the X-axis. Accordingly, the NIMPLY gate (with
strong RBS in OR gate component) in Fig. 4C shows that at 5.00E-05M arabinose and
28mM rhamnose, an RFP output of ~1775au was observed. That is, when pHrpL
promoter was used to drive the production of λCl repressors, the percentage
repression in the overall XOR logic was only ~37%, resulting in significant expression
leakiness.
% Repression =
=
IMPLYAbs,0M arabinose − IMPLYAbs,5.00E−05M arabinose
× 100
IMPLYAbs,0M arabinose
2830 − 1775
× 100 = 37%
2830
𝐄𝐪𝐧. (10)
From the above results, we reasoned that the OR gate that was originally constructed
with strong RBS should be replaced with a weaker RBS so that the layered NOT gate
will not be leaky. This fine-tuning procedure enabled the sequential layering of AND,
OR and NOT logic gates to display robust XOR digital operation in single (bacterial)
cell.
23
Sequence Information
BLUE: PROMOTER
GREEN: RBS
PURPLE: GENE CODING SEQUENCE
RED: TERMINATOR
BLACK: DNA SCAR
Input Switch Device A with strong RbsA (pSB1C3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pBAD.RbsA.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT
TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC
CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATT
TGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGC
TTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGAAAGAGGAGAAAATGGCTTCCTCCGAAG
ACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAA
ATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGG
TGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTA
AACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTT
ATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCAT
24
CTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGG
GTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGT
CTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGT
TCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCA
TCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTAGAGCCAGGCA
TCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACG
CTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
Input Switch Device A with weak RbsA (pSB1C3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pBAD.RbsB.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT
TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC
CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATT
TGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGC
TTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGTCACACAGGAAAGTACTAGATGGCTTCC
TCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGA
GTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTA
CCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCT
TACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGA
ACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTG
AGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAA
ACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAA
25
AATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAA
AACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGAC
TACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTAGAG
CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG
GTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
Input Switch Device B with strong RbsA (pSB1A3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pRHAB.RbsA.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCACAAT
TCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTA
ACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGAAAGAGGAGAAAATGGCT
TCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCA
CGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAG
TTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAA
GCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATG
GGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACG
GTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAA
AAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAAT
CAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTA
AAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAA
GACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTA
26
GAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
TCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
Input Switch Device B with weak RbsB (pSB1A3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pRHAB.RbsB.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCACAAT
TCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTA
ACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGTCACACAGGAAAGTACTA
GATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTA
ACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAA
CTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGG
TTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTT
TCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTG
CAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTAT
GCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAG
GTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTAC
ATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCA
CAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAAT
AATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTG
TTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTT
ATA
27
Pairwise Compatibility Test Construct (pSB1A3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pRHAB.RbsA.RFP.T15-pBAD.RbsA2.GFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCACAAT
TCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTA
ACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGAAAGAGGAGAAAATGGCT
TCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCA
CGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAG
TTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAA
GCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATG
GGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACG
GTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAA
AAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAAT
CAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTA
AAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAA
GACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTA
GAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
TCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACT
AGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTC
TCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGAC
AAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCA
CACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGCAA
CTCTCTACTGTTTCTCCATATACTAGAGGGAGCCCAATACTAGATGCGTAAAGGAGAAGAACTTTTCA
CTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGA
GAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACC
28
TGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATC
ATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTT
TTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAG
AATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACT
ATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATT
AGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGA
TGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAA
CTATACAAATAATAA
σ54 Promoter Genetic Context Effect Test (pSB1C3, pMB1 origin or pSB4A5, pSC101 origin)
pHrpL.Rbs.GFP.T15
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAGATTAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGT
CCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAG
GTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGG
CCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACA
GCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATG
ACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTA
AAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACA
CAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACA
TTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTC
CTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGA
CCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAT
AATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT
CTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG
TTTATA
pHrpL.Rbs.GFP.T15-pHrpL.Rbs.λCl.T15
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAGATTAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGT
CCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAG
GTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGG
CCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACA
GCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATG
ACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTA
AAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACA
CAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACA
TTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTC
CTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGA
CCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAT
AATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT
CTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG
TTTATATACTAGAGGCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAG
29
CTGACCGATGTTTTTGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAA
AGAATTTCACATTTTAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTA
TCGCTATAGGGCTTGTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAA
CACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGC
TTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGG
CATCAATGCATTAAATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAAT
TTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGA
AGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTT
TACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTG
AGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATT
CTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTT
TACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAA
TGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACG
TTTGGCTAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT
TTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTC
TGCGTTTATA
pHrpL.Rbs.λCl.T15-pHrpL.Rbs.GFP.T15
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTT
GAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATC
TGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAA
ATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATC
GCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATG
CGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAAT
TCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGA
GCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAAC
TGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAAT
GAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATACTA
GAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
TCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACT
AGAGGCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATG
TTTTTGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCAC
ATTTTAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGG
GCTTGTACTACTAGAGATTAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAG
TTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGT
GAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCC
ATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGA
AACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAA
GATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGA
GTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACT
CACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACAC
AACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCC
TGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGA
GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATAC
AAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT
TTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTC
TGCGTTTATA
30
Rbs.λCl.T15-pHrpL.Rbs.GFP.T15
AAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACG
TCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACA
AGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAAC
GCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAAT
CTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTT
CTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGG
GTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGC
ACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTG
AGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGAT
AGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTC
CGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATACTAGAGCCAGGCAT
CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGC
TCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGGCCGGAT
TATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTTTGTGCCA
AAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTTTAAAATA
TCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTTGTACTAC
TAGAGATTAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATT
CTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGC
AACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACAC
TTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGAC
TTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAA
CTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA
TTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTA
TACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGA
TGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTAC
CAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATG
GTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATA
CTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGT
TTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
pHrpL.Rbs.λCl.T15
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTT
GAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATC
TGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAA
ATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATC
GCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATG
CGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAAT
TCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGA
GCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAAC
TGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAAT
GAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATACTA
GAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
TCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
31
AND GATE (pSB4A5 plasmid, pSC101 replication origin)
pHrpL.Rbs.GFP.T15-pBAD.Rbs.HrpS.T15-pRhaB.Rbs.HrpR.T15-pCON.Rbs.RhaS.Rbs.AraC.T15
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAGATTAAAGAGGAGAAATACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGT
CCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAG
GTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGG
CCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACA
GCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATG
ACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTA
AAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACA
CAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACA
TTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTC
CTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGA
CCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAAT
AATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT
CTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG
TTTATATACTAGAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTAC
TGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAA
AGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGC
ACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTT
TTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGTCACACAGGACTACTAGATGACTATAATGAT
GAGTCTTGATGAAAGGTTTGAGGATGATCTGGACGAGGAGCGGGTTCCGAATCTGGGGATAGTTGCCG
AAAGTATTTCGCAACTGGGTATCGACGTGCTGCTATCGGGTGAGACCGGCACGGGCAAAGACACGATT
GCCCGACGGATTCATGAGATGTCAGGCCGCAAAGGGCGCCTGGTGGCGATGAATTGCGCGGCCATTCC
GGAGTCCCTCGCCGAGAGCGAGTTATTCGGCGTGGTCAGCGGTGCCTACACCGGCGCTGATCGCTCCA
GAGTCGGTTATGTCGAAGCGGCGCAGGGCGGCACGCTGTACCTGGATGAGATCGATAGCATGCCGCTG
AGCCTGCAAGCCAAATTGCTGAGGGTGCTGGAAACCCGAGCGCTTGAACGGCTGGGTTCGACGTCGAC
GATCAAGCTGGATATCTGCGTGATCGCCTCCGCCCAATGCTCGCTGGACGACGCCGTCGAGCGGGGGC
AGTTTCGTCGCGATCTGTATTTTCGCCTGAACGTCCTGACACTCAAGCTTCCTCCGCTACGTAACCAG
TCTGATCGCATAGTTCCCCTGTTCACACGTTTTACGGCCGCCGCCGCGAGGGAGCTCGGTGTTCCCGT
TCCCGATGTTTGCCCACTGCTGCACAAAGTGCTGCTGGGCCACGACTGGCCCGGCAATATCCGTGAGC
TCAAGGCGGCAGCCAAACGCCATGTGCTGGGTTTCCCCTTGCTGGGCGCCGAGCCGCAGGGCGAAGAG
CACTTGGCCTGTGGGCTCAAATCGCAATTGCGAGTGATCGAAAAAGCCCTGATTCAGGAGTCGCTCAA
GCGCCACGACAATTGTGTGGATTCGGTAAGCCTGGAACTGGACGTGCCACGCCGTACGCTCTATCGAC
GCATCAAAGAATTGCAGATCTAATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGG
CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGT
GGGCCTTTCTGCGTTTATATAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCA
CAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTC
AGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGTCACACAGGAAACC
TACTAGATGAGTACAGGCATCGATAAGGACGTCCGAGAGTGTTGGGGCGTAACTGCATTATCAGCGGG
TCATCAAATTGCAATGAATAGCGCGTTTCTGGATATGGACTTGCTGTTGTGCGGGGAAACCGGCACCG
GCAAGGACACACTGGCCAACCGCATTCACGAGTTGTCCAGCAGGTCGGGACCCTTTGTGGGCATGAAC
TGCGCCGCCATTCCCGAGTCGCTGGCAGAGAGCCAGTTATTCGGTGTGGTCAACGGTGCATTCACCGG
CGTATGCCGGGCTCGCGAGGGCTACATAGAGGCCTCCAGTGGTGGCACCTTGTACCTGGATGAAATCG
ACAGCATGCCGTTGAGCCTGCAAGCCAAACTGCTGCGTGTGTTGGAGAGTCGAGGTATCGAGCGTCTG
GGCTCGACCGAATTTATCCCGGTGGATCTGCGGATCATTGCCTCGGCCCAGCGGCCACTGGATGAACT
GGTGGAACAAGGACTTTTCCGTCGCGACCTGTTTTTTCGGCTCAACGTGCTGACGCTTCACTTGCCAG
CCTTGCGCAAACGTCGTGAACAGATCCTGCCATTGTTCGACCAGTTCACCCAGGGTATCGCTGCCGAG
TTCGGACGTCCCGCTCCTGCGCTGGACAGCGGGCGTGTGCAGCTGCTGCTCAGCCACGACTGGCCGGG
CAACATCCGCGAATTGAAGTCTGCGGCCAAGCGCTTCGTACTCGGCTTCCCCTTGCTGGGCGCCGACC
32
CTGTGGAAGCGCTTGACCCTGCCACGGGGCTGCGCACGCAAATGCGCATCATCGAGAAAATGCTCATC
CAGGATGCCTTGAAGCGGCACAGGCACAATTTCGACGCGGTGCTTCAGGAGTTGGAGTTGCCAAGACG
CACCCTGTATCACCGCATGAAGGAACTGGGAGTTGCAGCGCCGATCGCTGCGACGGCCGGGGTCTAAT
AACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGT
CGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTA
GATTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACA
TAGTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATT
TTCCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTT
AATGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTA
TGAACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCG
CCGGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCAC
AGCGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACC
CTCGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGG
AGAACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGAT
GAGGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAA
GCAGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGC
TACGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTT
TCGACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCT
GCAATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTA
ACGCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGA
CCGCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGG
ACGAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTC
GTCATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAA
TGGCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCA
TTTCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGG
CGATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCG
ATGGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATAT
CGCCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAG
GGATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACC
CGGATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATT
TAAAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAG
CCGTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT
TTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTC
TGCGTTTATA
33
OR GATE I (pSB1C3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pBAD-pRHAB.RbsA.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT
TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC
CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATT
TGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGC
TTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGAGACCTTTACGCCGCTGGAGCAGGAATG
CGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGC
CAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATA
CTAGAGAAAGAGGAGAAAATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCG
TATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAG
GTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCC
CCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACT
GTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTA
CCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCG
TCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGA
AGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTG
AAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAA
CTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCA
CTCCACCGGTGCTTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGG
GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGG
TGGGCCTTTCTGCGTTTATA
34
OR GATE II (pSB1C3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pRHAB-pBAD.RbsA.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCACAAT
TCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTA
ACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGAGAAGAAACCAATTGTCC
ATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCC
CGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTC
TATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATT
TTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATAAA
AGAGGAGAAAATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAG
GTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAG
ACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTT
CCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCC
CGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGAC
TCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGG
TCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTG
CTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAA
ACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACAT
CACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCG
GTGCTTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCG
TTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTT
TCTGCGTTTATA
35
OR GATE III (pSB1C3, pMB1 replication origin)
pCON.Rbs.RhaS.Rbs.AraC.T15-pBAD.RbsA.RFP.T15-pRHAB.RbsA.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT
TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC
CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATT
TGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGC
TTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGAAAGAGGAGAAAATGGCTTCCTCCGAAGA
CGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAA
TCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGT
GGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAA
ACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTA
TGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATC
TACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGG
TTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTC
TGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTT
CAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCAT
CGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTAGAGCCAGGCATCA
AATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTA
GAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGAGACCTTTACGCCGCTGGAG
CAGGAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCC
CTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTG
GTCGTATACTAGAGAAAGAGGAGAAAATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCA
AAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCG
TACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACAT
36
CCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACC
TGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTT
ACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAA
CTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGT
ACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTAC
GACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGA
CATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAG
GTCGTCACTCCACCGGTGCTTAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGAC
TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGG
CCTTTCTGCGTTTATA
37
NIMPLY GATE (pSB1C3, pMB1 replication origin)
*TATCACCGGGCGTGCTGATA palindromic λCl binding sites
pCON.RbsRhaS.Rbs.AraC.T15.pBAD.pRHABCl2B.RbsA.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT
TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC
CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATT
TGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGC
TTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAAAAGAGGAGAAATACTAGATGAGCACAAA
AAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGA
AAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGT
GCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGT
TAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGC
AGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCT
GAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTC
TGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTG
ACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTT
GGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAA
CCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGT
GGCCTGAAGAGACGTTTGGCTAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGA
CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTT
CGGGTGGGCCTTTCTGCGTTTATAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACT
38
GCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAA
AGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACAT
TGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTA
CCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGAGACCTTTACGCCGCTGGAG
CAGGAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCC
CTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTG
GTCGTATATCACCGGGCGTGCTGATACAGTCATACTAGAAAAGAGGAGAAAATGGCTTCCTCCGAAGA
CGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAA
TCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGT
GGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAA
ACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTA
TGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATC
TACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGG
TTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTC
TGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTT
CAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCAT
CGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTAGAGCCAGGCATCA
AATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTA
GAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
pCON.RbsRhaS.Rbs.AraC.T15.pBAD.pRHABCl2B.RbsB.RFP.T15
TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATA
GTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTT
CCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAA
TGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATG
AACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCC
GGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG
CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCT
CGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAG
AACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGA
GGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGC
AGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTA
CGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTC
GACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGC
AATAACGAGAGAGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAAC
GCCCATCTGGTGGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACC
GCTGGGAATGAAAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGAC
GAGAATTTGTCTGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGT
CATCCGGAGGCTCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATG
GCTTAACTGGCCGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATT
TCAGCGACCTGTTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCG
ATAAATCTGCTTGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGAT
GGATAATCGGGTACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCG
CCAGCGTCGCACAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGG
ATTAGCGTCTTAAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCG
GATGCCTATCGCCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTA
AAAAATGCACCGGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCC
GTCAAGTTGTCATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT
ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTG
CGTTTATATACTAGAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTT
TACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGAC
39
CAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATT
TGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGC
TTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAAAAGAGGAGAAATACTAGATGAGCACAAA
AAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGA
AAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGT
GCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGT
TAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGC
AGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCT
GAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTC
TGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTG
ACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTT
GGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAA
CCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGT
GGCCTGAAGAGACGTTTGGCTAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGA
CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTT
CGGGTGGGCCTTTCTGCGTTTATAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACT
GCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAA
AGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACAT
TGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTA
CCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATATACTAGAGAGACCTTTACGCCGCTGGAG
CAGGAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCC
CTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTG
GTCGTATATCACCGGGCGTGCTGATACAGTCATACTAGATCACACAGGAAAGTACTAGATGGCTTCCT
CCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAG
TTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTAC
CAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTT
ACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAA
CGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGA
GTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAA
CCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAA
ATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAA
ACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACT
ACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAATACTAGAGCC
AGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCT
CTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
40
XOR GATE (AND GATE in pSB4A5 & OR-NOT GATE in pSB1C3)
pBAD.Rbs.HrpS.T15-pRhaB.Rbs.HrpR.T15-pCON.Rbs.RhaS.Rbs.AraC.T15 [pSB4A5]
AGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGC
TAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAA
ACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACT
TTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGCAACTCT
CTACTGTTTCTCCATATACTAGAGTCACACAGGACTACTAGATGACTATAATGATGAGTCTTGATGAA
AGGTTTGAGGATGATCTGGACGAGGAGCGGGTTCCGAATCTGGGGATAGTTGCCGAAAGTATTTCGCA
ACTGGGTATCGACGTGCTGCTATCGGGTGAGACCGGCACGGGCAAAGACACGATTGCCCGACGGATTC
ATGAGATGTCAGGCCGCAAAGGGCGCCTGGTGGCGATGAATTGCGCGGCCATTCCGGAGTCCCTCGCC
GAGAGCGAGTTATTCGGCGTGGTCAGCGGTGCCTACACCGGCGCTGATCGCTCCAGAGTCGGTTATGT
CGAAGCGGCGCAGGGCGGCACGCTGTACCTGGATGAGATCGATAGCATGCCGCTGAGCCTGCAAGCCA
AATTGCTGAGGGTGCTGGAAACCCGAGCGCTTGAACGGCTGGGTTCGACGTCGACGATCAAGCTGGAT
ATCTGCGTGATCGCCTCCGCCCAATGCTCGCTGGACGACGCCGTCGAGCGGGGGCAGTTTCGTCGCGA
TCTGTATTTTCGCCTGAACGTCCTGACACTCAAGCTTCCTCCGCTACGTAACCAGTCTGATCGCATAG
TTCCCCTGTTCACACGTTTTACGGCCGCCGCCGCGAGGGAGCTCGGTGTTCCCGTTCCCGATGTTTGC
CCACTGCTGCACAAAGTGCTGCTGGGCCACGACTGGCCCGGCAATATCCGTGAGCTCAAGGCGGCAGC
CAAACGCCATGTGCTGGGTTTCCCCTTGCTGGGCGCCGAGCCGCAGGGCGAAGAGCACTTGGCCTGTG
GGCTCAAATCGCAATTGCGAGTGATCGAAAAAGCCCTGATTCAGGAGTCGCTCAAGCGCCACGACAAT
TGTGTGGATTCGGTAAGCCTGGAACTGGACGTGCCACGCCGTACGCTCTATCGACGCATCAAAGAATT
GCAGATCTAATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT
CTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG
TTTATATAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCACAATTCAGCAAAT
TGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGG
TCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGTCACACAGGAAACCTACTAGATGAGTA
CAGGCATCGATAAGGACGTCCGAGAGTGTTGGGGCGTAACTGCATTATCAGCGGGTCATCAAATTGCA
ATGAATAGCGCGTTTCTGGATATGGACTTGCTGTTGTGCGGGGAAACCGGCACCGGCAAGGACACACT
GGCCAACCGCATTCACGAGTTGTCCAGCAGGTCGGGACCCTTTGTGGGCATGAACTGCGCCGCCATTC
CCGAGTCGCTGGCAGAGAGCCAGTTATTCGGTGTGGTCAACGGTGCATTCACCGGCGTATGCCGGGCT
CGCGAGGGCTACATAGAGGCCTCCAGTGGTGGCACCTTGTACCTGGATGAAATCGACAGCATGCCGTT
GAGCCTGCAAGCCAAACTGCTGCGTGTGTTGGAGAGTCGAGGTATCGAGCGTCTGGGCTCGACCGAAT
TTATCCCGGTGGATCTGCGGATCATTGCCTCGGCCCAGCGGCCACTGGATGAACTGGTGGAACAAGGA
CTTTTCCGTCGCGACCTGTTTTTTCGGCTCAACGTGCTGACGCTTCACTTGCCAGCCTTGCGCAAACG
TCGTGAACAGATCCTGCCATTGTTCGACCAGTTCACCCAGGGTATCGCTGCCGAGTTCGGACGTCCCG
CTCCTGCGCTGGACAGCGGGCGTGTGCAGCTGCTGCTCAGCCACGACTGGCCGGGCAACATCCGCGAA
TTGAAGTCTGCGGCCAAGCGCTTCGTACTCGGCTTCCCCTTGCTGGGCGCCGACCCTGTGGAAGCGCT
TGACCCTGCCACGGGGCTGCGCACGCAAATGCGCATCATCGAGAAAATGCTCATCCAGGATGCCTTGA
AGCGGCACAGGCACAATTTCGACGCGGTGCTTCAGGAGTTGGAGTTGCCAAGACGCACCCTGTATCAC
CGCATGAAGGAACTGGGAGTTGCAGCGCCGATCGCTGCGACGGCCGGGGTCTAATAACCAGGCATCAA
ATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCT
CTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGATTTACAGCTAG
CTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATAGTGTGGATTTT
TTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTTCCTGAACATCA
TCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAATGGGCAGCCCT
ATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATGAACATACCGAT
AATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCCGGGCTGAATCA
GTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAGCGTATTGCAGC
AGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCTCGACCGCCAGT
CGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAGAACCTGGAAAA
CAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGAGGTGAATTGGG
ATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGCAGCAAACGGGA
CTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTACGCCACAGCGA
41
GGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTCGACGCTTTTTC
GCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGCAATAACGAGAG
AGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAACGCCCATCTGGT
GGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACCGCTGGGAATGA
AAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGACGAGAATTTGTC
TGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGTCATCCGGAGGC
TCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATGGCTTAACTGGC
CGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATTTCAGCGACCTG
TTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCGATAAATCTGCT
TGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGATGGATAATCGGG
TACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCGCCAGCGTCGCA
CAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGGATTAGCGTCTT
AAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCGGATGCCTATCG
CCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTAAAAAATGCACC
GGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCCGTCAAGTTGTC
ATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTT
GTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
XOR GATE I
pHrpL.Rbs.λcl.T15-pBAD.pRHABCl2B.RbsB.RFPasv.T15 [pSB1C3]
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTT
GAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATC
TGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAA
ATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATC
GCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATG
CGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAAT
TCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGA
GCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAAC
TGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAAT
GAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATAATA
CTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGT
TTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAT
ACTAGAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT
TCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATG
ACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGT
CACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGC
AACTCTCTACTGTTTCTCCATATACTAGACAGGAATGCGGTGAGCATCACATCACCACAATTCAGCAA
ATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAA
GGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATATCACCGGGCGTGCTGATACAGTCATACTAGA
TCACACAGGAAAGTACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTC
GTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAA
GGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTC
CCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAAC
TGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTT
ACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCC
GTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGG
42
AAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCT
GAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAA
ACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTC
ACTCCACCGGTGCTGCTGCAAACGACGAAAACTACGCTGCATCTGTATAATACTAGAGCCAGGCATCAAA
TAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGA
GTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
XOR GATE II
pHrpL.Rbs.λcl.T15-pBADCl2B.pRHABCl2B.RbsB.RFPasv.T15 [pSB1C3]
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTT
GAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATC
TGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAA
ATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATC
GCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATG
CGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAAT
TCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGA
GCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAAC
TGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAAT
GAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATAATA
CTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGT
TTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAT
ACTAGAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT
TCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATG
ACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGT
CACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGC
AACTCTCTACTGTTTCTCCATATATCACCGGGCGTGCTGATATACTAGACAGGAATGCGGTGAGCATC
ACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCAT
TTTCCTGTCAGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATATCACCGGGCGT
GCTGATACAGTCATACTAGATCACACAGGAAAGTACTAGATGGCTTCCTCCGAAGACGTTATCAAAGA
GTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAG
GTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCG
TTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGA
CATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAG
ACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAA
CTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTC
CACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAG
ACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGT
GCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTA
CGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTGCTGCAAACGACGAAAACTACGCTGCATCTGTAT
AATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTG
TCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
43
XOR GATE II
pHrpL.Rbs.λcl.T15-pBADCl2B.pRHABCl2B.RbsB.RFP.T15 [pSB1C3]
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTT
GAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATC
TGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAA
ATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATC
GCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATG
CGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAAT
TCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGA
GCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAAC
TGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAAT
GAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATAATA
CTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGT
TTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAT
ACTAGAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT
TCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATG
ACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGT
CACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGC
AACTCTCTACTGTTTCTCCATATATCACCGGGCGTGCTGATATACTAGACAGGAATGCGGTGAGCATC
ACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCAT
TTTCCTGTCAGTAACGAGAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATATCACCGGGCGT
GCTGATACAGTCATACTAGATCACACAGGAAAGTACTAGATGGCTTCCTCCGAAGACGTTATCAAAGA
GTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAG
GTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCG
TTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGA
CATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAG
ACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAA
CTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTC
CACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAG
ACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGT
GCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTA
CGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCT
CAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCA
CCTTCGGGTGGGCCTTTCTGCGTTTATA
XOR GATE IV
pHrpL.Rbs.λcl.T15-pBADCl2B.RbsB.RFPasv.T15-pRHABCl2B.RbsB.RFPasv.T15 [pSB1C3]
GCCGGATTATGTCCGCTGAGTGGGTCACGGTCCCGGATCAGTTCCCTTGCGAAGCTGACCGATGTTTT
TGTGCCAAAAGCTGTTGTGGCAAAAAACGGTTTGCGCAAAGTTTTGTATTACAAAGAATTTCACATTT
TAAAATATCTTTATAAATCAATCAGTTATTTCTATTTTTAAGCTGGCATGGTTATCGCTATAGGGCTT
GTACTACTAGAAAAGAGGAGAAATACTAGATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTT
GAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATC
TGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAA
ATGCTTATAACGCCGCATTGCTTGCAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATC
GCCAGAGAAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
44
CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTGAGCTTAGAACCTTTACCAAAGGTGATG
CGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAAT
TCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGA
GCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAAC
TGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAAT
GAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTAATAATA
CTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGT
TTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAT
ACTAGAAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCT
TCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATG
ACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGT
CACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGC
AACTCTCTACTGTTTCTCCATATATCACCGGGCGTGCTGATACAGTCATACTAGATCACACAGGAAAG
TACTAATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCC
GTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGC
TAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGT
ACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAA
GGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTC
CCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGG
TTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTG
AAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCAC
CTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCT
CCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCT
GCTGCAAACGACGAAAACTACGCTGCATCTGTATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTC
AGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCAC
CTTCGGGTGGGCCTTTCTGCGTTTATATACTAGACAGGAATGCGGTGAGCATCACATCACCACAATTCAG
CAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGA
GAAGGTCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATATCACCGGGCGTGCTGATACAGTCATACT
AGATCACACAGGAAAGTACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAG
TTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTAC
GAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCT
GTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGA
AACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACC
GTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTT
CCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACC
CGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGAC
GCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACAT
CAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTC
GTCACTCCACCGGTGCTGCTGCAAACGACGAAAACTACGCTGCATCTGTATAATACTAGAGCCAGGCAT
CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTAC
TAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
45
HALF ADDER
The HALF ADDER was created using DNA sequences of AND GATE and XOR GATE IV as described above.
HALF SUBTRACTOR
The HALF SUBTRACTOR was created using DNA sequences of plasmid pAW-SUB and XOR GATE IV.
pBAD.Rbs.HrpS.T15-pRHAB.Rbs.HrpR.T15-pCON.Rbs.RhaS.Rbs.AraC.T15-pBADCl2B.Rbs.GFP.T15
[Designated pAW-SUB, pSB4A5]
AGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGC
TAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAA
ACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACT
TTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGCAACTCT
CTACTGTTTCTCCATATACTAGAGTCACACAGGACTACTAGATGACTATAATGATGAGTCTTGATGAA
AGGTTTGAGGATGATCTGGACGAGGAGCGGGTTCCGAATCTGGGGATAGTTGCCGAAAGTATTTCGCA
ACTGGGTATCGACGTGCTGCTATCGGGTGAGACCGGCACGGGCAAAGACACGATTGCCCGACGGATTC
ATGAGATGTCAGGCCGCAAAGGGCGCCTGGTGGCGATGAATTGCGCGGCCATTCCGGAGTCCCTCGCC
GAGAGCGAGTTATTCGGCGTGGTCAGCGGTGCCTACACCGGCGCTGATCGCTCCAGAGTCGGTTATGT
CGAAGCGGCGCAGGGCGGCACGCTGTACCTGGATGAGATCGATAGCATGCCGCTGAGCCTGCAAGCCA
AATTGCTGAGGGTGCTGGAAACCCGAGCGCTTGAACGGCTGGGTTCGACGTCGACGATCAAGCTGGAT
ATCTGCGTGATCGCCTCCGCCCAATGCTCGCTGGACGACGCCGTCGAGCGGGGGCAGTTTCGTCGCGA
TCTGTATTTTCGCCTGAACGTCCTGACACTCAAGCTTCCTCCGCTACGTAACCAGTCTGATCGCATAG
TTCCCCTGTTCACACGTTTTACGGCCGCCGCCGCGAGGGAGCTCGGTGTTCCCGTTCCCGATGTTTGC
CCACTGCTGCACAAAGTGCTGCTGGGCCACGACTGGCCCGGCAATATCCGTGAGCTCAAGGCGGCAGC
CAAACGCCATGTGCTGGGTTTCCCCTTGCTGGGCGCCGAGCCGCAGGGCGAAGAGCACTTGGCCTGTG
GGCTCAAATCGCAATTGCGAGTGATCGAAAAAGCCCTGATTCAGGAGTCGCTCAAGCGCCACGACAAT
TGTGTGGATTCGGTAAGCCTGGAACTGGACGTGCCACGCCGTACGCTCTATCGACGCATCAAAGAATT
GCAGATCTAATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTAT
CTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG
TTTATATAGACCTTTACGCCGCTGGAGCAGGAATGCGGTGAGCATCACATCACCACAATTCAGCAAAT
TGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGG
TCGCGAATTGAGGCGCTTTTTAGACTGGTCGTATACTAGAGTCACACAGGAAACCTACTAGATGAGTA
CAGGCATCGATAAGGACGTCCGAGAGTGTTGGGGCGTAACTGCATTATCAGCGGGTCATCAAATTGCA
ATGAATAGCGCGTTTCTGGATATGGACTTGCTGTTGTGCGGGGAAACCGGCACCGGCAAGGACACACT
GGCCAACCGCATTCACGAGTTGTCCAGCAGGTCGGGACCCTTTGTGGGCATGAACTGCGCCGCCATTC
CCGAGTCGCTGGCAGAGAGCCAGTTATTCGGTGTGGTCAACGGTGCATTCACCGGCGTATGCCGGGCT
CGCGAGGGCTACATAGAGGCCTCCAGTGGTGGCACCTTGTACCTGGATGAAATCGACAGCATGCCGTT
GAGCCTGCAAGCCAAACTGCTGCGTGTGTTGGAGAGTCGAGGTATCGAGCGTCTGGGCTCGACCGAAT
TTATCCCGGTGGATCTGCGGATCATTGCCTCGGCCCAGCGGCCACTGGATGAACTGGTGGAACAAGGA
CTTTTCCGTCGCGACCTGTTTTTTCGGCTCAACGTGCTGACGCTTCACTTGCCAGCCTTGCGCAAACG
TCGTGAACAGATCCTGCCATTGTTCGACCAGTTCACCCAGGGTATCGCTGCCGAGTTCGGACGTCCCG
CTCCTGCGCTGGACAGCGGGCGTGTGCAGCTGCTGCTCAGCCACGACTGGCCGGGCAACATCCGCGAA
TTGAAGTCTGCGGCCAAGCGCTTCGTACTCGGCTTCCCCTTGCTGGGCGCCGACCCTGTGGAAGCGCT
TGACCCTGCCACGGGGCTGCGCACGCAAATGCGCATCATCGAGAAAATGCTCATCCAGGATGCCTTGA
AGCGGCACAGGCACAATTTCGACGCGGTGCTTCAGGAGTTGGAGTTGCCAAGACGCACCCTGTATCAC
CGCATGAAGGAACTGGGAGTTGCAGCGCCGATCGCTGCGACGGCCGGGGTCTAATAACCAGGCATCAA
ATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCT
CTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGATTTACAGCTAG
CTCAGTCCTAGGTATTATGCTAGCTACTAGAGGGAGCCCAAATGACCGTATTACATAGTGTGGATTTT
TTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTTCCTGAACATCA
TCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAATGGGCAGCCCT
46
ATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATGAACATACCGAT
AATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCCGGGCTGAATCA
GTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAGCGTATTGCAGC
AGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCTCGACCGCCAGT
CGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAGAACCTGGAAAA
CAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGAGGTGAATTGGG
ATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGCAGCAAACGGGA
CTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTACGCCACAGCGA
GGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTCGACGCTTTTTC
GCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGCAATAACGAGAG
AGAGTTAACATGGCTGAAGCGCAAAATGATCCCCTGCTGCCGGGATACTCGTTTAACGCCCATCTGGT
GGCGGGTTTAACGCCGATTGAGGCCAACGGTTATCTCGATTTTTTTATCGACCGACCGCTGGGAATGA
AAGGTTATATTCTCAATCTCACCATTCGCGGTCAGGGGGTGGTGAAAAATCAGGGACGAGAATTTGTC
TGCCGACCGGGTGATATTTTGCTGTTCCCGCCAGGAGAGATTCATCACTACGGTCGTCATCCGGAGGC
TCGCGAATGGTATCACCAGTGGGTTTACTTTCGTCCGCGCGCCTACTGGCATGAATGGCTTAACTGGC
CGTCAATATTTGCCAATACGGGTTTCTTTCGCCCGGATGAAGCGCACCAGCCGCATTTCAGCGACCTG
TTTGGGCAAATCATTAACGCCGGGCAAGGGGAAGGGCGCTATTCGGAGCTGCTGGCGATAAATCTGCT
TGAGCAATTGTTACTGCGGCGCATGGAAGCGATTAACGAGTCGCTCCATCCACCGATGGATAATCGGG
TACGCGAGGCTTGTCAGTACATCAGCGATCACCTGGCAGACAGCAATTTTGATATCGCCAGCGTCGCA
CAGCATGTTTGCTTGTCGCCGTCGCGTCTGTCACATCTTTTCCGCCAGCAGTTAGGGATTAGCGTCTT
AAGCTGGCGCGAGGACCAACGCATTAGTCAGGCGAAGCTGCTTTTGAGCACTACCCGGATGCCTATCG
CCACCGTCGGTCGCAATGTTGGTTTTGACGATCAACTCTATTTCTCGCGAGTATTTAAAAAATGCACC
GGGGCCAGCCCGAGCGAGTTTCGTGCCGGTTGTGAAGAAAAAGTGAATGATGTAGCCGTCAAGTTGTC
ATAACCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTT
GTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAAGA
AGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAA
CCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACG
CGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTG
CTATGCCATAGCATTTTTATCCATAAGATTAGCGGATTCTACCTGACGCTTTTTATCGCAACTCTCTA
CTGTTTCTCCATATATCACCGGGCGTGCTGATACAGTCATACTAGATTAAAGAGGAGAAATACTAGAT
GCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATG
GGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTT
ATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCA
ATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTT
ATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTT
GAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCT
TGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATG
GAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTAT
CAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATC
TGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTG
GGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAA
GGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTC
ACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
References
1.
Wang BJ, Kitney RI, Joly N, Buck M: Engineering modular and orthogonal genetic logic gates
for robust digital-like synthetic biology. Nature Communications 2011, 2.
47