S1

Supporting Information

Generation of an Orthogonal Protein–Protein Interface with a Noncanonical Amino Acid

Minseob Koh,† Fariborz Nasertorabi,

‡ Gye Won Han,

‡ Raymond C. Stevens,

‡* and Peter G. Schultz

†*

†Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N Torrey Pines

Road, La Jolla, California 92037, United States ‡Department of Biological Sciences, Bridge Institute, University of Southern California, 3430 S Vermont Avenue, Los Ange-

les, California 90089, United States

Materials and Methods

Strain and reagents. The chorismate mutase deficient E. coli strain KA12/pKIMP-UAUC was a gift from Dr.

Donald Hilvert (ETH, Zürich, Switzerland).1 XL1-Blue was obtained from Agilent Technologies Inc. (La Jolla,

CA). DH10B and BL21(DE3) were obtained from ThermoFisher Scientific (Waltham, MA). Restriction enzymes

NdeI, XhoI, KpnI-HF, SphI-HF, BglII, BamHI-HF, DpnI, CIP and T4 DNA ligase were purchased from New

England BioLabs (NEB, Ipswich, MA). Synthetic oligonucleotides were purchased from Integrated DNA Tech-

nologies (IDT, San Diego, CA). PfuUltra II Fusion HS DNA polymerase and PfuTurbo DNA polymerase (Agilent

Technologies Inc.) were used for general PCR experiments and site directed mutagenesis, respectively. PCR

products and products of restriction digestion were purified by agarose gel electrophoresis, Zymoclean Gel DNA

Recovery kit and DNA Clean & Concentrator (Zymo Research, Irvine, CA). Plasmid DNA was purified by ZR

Plasmid Miniprep kit (Zymo Research, Irvine, CA). DNA sequence analysis was performed by Genewiz (La Jol-

la, CA). Chorismic acid was prepared as previously described.2 p-benzoyl phenylalanine was purchased from

Combi-Blocks, Inc. (San Diego, CA).

Figure S1. Metabolic pathway for tyrosine and phenylalanine biosynthesis in chorismate mutase knock out strain, KA12/pKIMP-UAUC.

Erwinia herbicola prephenate dehydrogenase and Pseudomonas aeruginosa prephenate dehydratase expressed from pKIMP-UAUC allow

prephenate bioconversion to generate tyrosine and phenylalanine.

S2

Plasmid construction. The ColE1 replicon, kanamycin resistance cassette of pKTECM was derived from pBK.3

The E. coli chorismate mutase gene that encodes the first 92 amino acids (Met1 to Leu92) of pheA gene was am-

plified from the DH10B total DNA with primers MK.01 and MK.02. The PCR product was digested with NdeI

and XhoI, and then ligated to the NdeI and XhoI fragment of pET-22b(+) (EMD Millipore, San Diego, CA) to

give pET-CM. The chorismate mutase gene with a C-terminal histidine tag was amplified with primers MK.01

and MK.03. The PCR product was digested with NdeI and KpnI-HF and then ligated with the NdeI and KpnI

fragment of pBK to give pBK-CM. The tetR gene and the Ptet promoter cassettes were cloned from XL1-Blue

total DNA with primers MK.04 and MK.05. The PCR product was digested with SphI-HF and BglII, then ligated

to the SphI-HF and BglII fragment of pET-22b(+) to give pET-TET. The NdeI site was silenced by site directed

mutagenesis with primers MK.06 and MK.07. The gene was amplified again with primers MK.08 and MK.09.

The PCR product was digested with BamHI-HF and NdeI, then ligated with the BamHI-HF and NdeI fragment of

pBK-CM to give pKTECM. The mutant plasmids pKTECM-Y72A and pET-CM-Y72A were constructed with the

primer pair MK.10/11, and pET-CM-Y72X was constructed with the primer pair MK.12/13.

To construct pUltra-BzF, the backbone of pUltra-Poly4 was amplified with primers MK.14 and MK.15 to

afford the Ultra-frag where contains the Clodf13 replicon, spectinomycin resistance cassette, lacI gene and

tRNACUA gene. The aaRS gene was amplified from pEVOL-pBpF5,6 with primers MK.16 and MK.17 to give

BzFRS-frag. Then, Ultra-frag and pBzFRS-frag were ligated by using Gibson Assembly Master Mix (NEB, MA)

to give pUltra-BzF.

Chorismate mutase library construction. To construct the chorismate mutase library, ultramers MK.18 and

MK.19 were annealed before PCR with primers MK.01 and MK.03. The PCR product was digested with NdeI

and KpnI-HF, and then ligated with the NdeI and KpnI-HF fragment of pKTECM. The ligated product was puri-

fied by precipitation with NaOAc (85 mM, pH 5.1), yeast tRNA (30 ng/μl, ThermoFisher Scientific) and ethanol

(70%) to give pKTECM-Y72X-NNK5 which contains the amber mutation at Y72 and NNK (N=A, C, G or T;

K=G or T) mutations at the residues Leu25’, Arg29’, Leu76, Ile80’ and Asp83’. Plasmid DNA was electroporated

into DH10B cells; more than 108 colonies were generated to ensure complete coverage of the library. Supercoiled

library plasmid was purified by NucleoBond Xtra Maxi (Macherey-Nagel Inc., Bethlehem, PA). Ec.ΔCM.BzF

cells were prepared by transforming electrocompetent KA12/pKIMP-UAUC cells with pUltra-BzF. pKTECM-

Y72X-NNK5 was electroporated into Ec.ΔCM.BzF cells, rescued with SOC medium, then plated onto LB agar

plates containing chloramphenicol (30 μg/ml), kanamycin (50 μg/ml) and spectinomycin (50 μg/ml), followed by

growth overnight at 30 °C. Library diversity was calculated by dilution plating and was determined to exceed 108.

Bacterial cells were harvested and stored as a glycerol stock.

Figure S2. Functional assay for mutants of Tyr72 in EcCM. (A) Complementation experiments with the wild type and Y72A variant

EcCM. The dilution series (10 fold dilutions from approximately 105 cells) of KA12/pKIMP-UAUC cells harboring pKTECM or

pKTECM-Y72A were spotted onto LB, M9c.FY and M9c agar plates and incubated at 30 °C for 3 days. Growth on LB plate was captured

at day 1. (B) Catalytic activity of wild type, Y72A and Y72pBzF (ECA) variants. Catalytic efficiency (kcat/Km) was calculated based on

Michaelis-Menten kinetics for the conversion of the chorismate to prephenate. Ni-affinity column purified non-homogeneous wild type

(WTc, closed circle), Y72A (open triangle) and Y72pBzF variant (ECA, closed diamond) were used. Error bars represent standard devia-

tion of triplicates.

S3

Figure S3. The aminoacyl-tRNA synthetase (aaRS)/tRNA pair expressed by pUltra-BzF specifically inserted pBzF into sfGFP at permis-

sive site 151. The DH10B/pET22b-T5-sfGFP*/pUltra-BzF cells were incubated under IPTG (1 mM) induction conditions in the presence

or absence of pBzF (1 mM), and sfGFP fluorescence was recorded. The data was normalized to the fluorescence value of the cells cultured

without pBzF.

Culture and selection condition. Selection medium was modified from previously reported M9c glucose mini-

mal medium.7 M9c minimal medium contains 6 mg/ml Na2HPO4, 3 mg/ml KH2PO4, 1 mg/ml NH4Cl, 0.5 mg/ml

NaCl, 0.2% (w/v) D-(+)-glucose, 1 mM MgSO4, 0.1 mM CaCl2, 5 μg/ml thiamine HCl, 5 μg/ml 4-hydroxybenzoic

acid, 5 μg/ml 4-aminobenzoic acid, 1.6 μg/ml 2,3-dihydroxybenzoic acid, and 20 μg/ml L-tryptophan. For plates,

agar (15 g/l) was added. L-phenylalanine (20 μg/ml) and L-tyrosine (20 μg/ml) were added for non-selective con-

trol experiments (M9c.FY).

The chorismate mutase-deficient strain KA12/pKIMP-UAUC was transformed either with pKTECM or

pKTECM-Y72A. After three washes with M9c medium, a dilution series of transformants (approximately 105,

104, 103 and 102 cells) was spotted onto the LB agar plates, M9c supplemented with tetracycline (100 ng/ml) or

non-selective control M9c.FY, respectively. Chloramphenicol (30 μg/ml) and kanamycin (50 μg/ml) were added,

and the plates were incubated at 30 °C for 3 days (Figure S2).

The activity of pUltra-BzF was determined by measuring the fluorescence of green fluorescence protein

(GFP). The DH10B transformant containing pET22b-T5-sfGFP*8 and pUltra-BzF was cultured in LB in the pres-

ence or absence of 1 mM pBzF at 30 °C with shaking at 250 rpm. After overnight induction with the isopropyl 1-

thio-β-D-galactopyranoside (IPTG, 1 mM), superfolder GFP (sfGFP) fluorescence was measured (Figure S3).

The pool of Ec.ΔCM.BzF cells containing the pKTECM-Y72X-NNK5 library was washed three times with M9c

medium before inoculating into M9c medium supplemented with pBzF (1 mM), IPTG (1 mM) and tetracycline

(100 ng/ml). Antibiotics required for plasmid maintenance were chloramphenicol (30 μg/ml), kanamycin (50

μg/ml) and spectinomycin (50 μg/ml). The starting OD600 was 0.03 with 30 mL medium in a 250 mL Erlenmeyer

flask. Growth was maintained until saturated at 30 °C with shaking at 250 rpm. The surviving clones were re-

plated onto LB agar plate containing chloramphenicol (30 μg/ml), kanamycin (50 μg/ml) and spectinomycin (50

μg/ml). 20 colonies were isolated and sequenced, and the hit clone pKTECB was identified. The growth rate of

Ec.ΔCM.BzF/pKTECB was compared with Ec.ΔCM.BzF/pKTECM in the presence or absence of pBzF (1 mM)

in M9c medium supplemented with IPTG (1 mM) and tetracycline (100 ng/ml). Starting OD600 was 0.03 with 30

mL M9c medium in a 250 mL Erlenmeyer flask. Growth was maintained for 6 days at 30 °C with shaking at 250

rpm and OD600 was monitored.

S4

Table S1. Growth analysis of KA12/pKIMP-UAUC cells harboring pKTECB-Y72X20

pKTECB-Y72X20 pKTECM

Substitution (X) Day 0 Day 10 Day 0 Day 10

G 0.03 0.02

0.03 0.89

A 0.03 0.02

S 0.03 0.02

T 0.03 0.03

C 0.03 0.02

V 0.03 0.03

L 0.03 0.03

I 0.03 0.03

M 0.03 0.03

P 0.03 0.02

F 0.03 0.03

Y 0.03 0.02

W 0.03 0.02

D 0.03 0.01

E 0.03 0.03

N 0.03 0.03

Q 0.03 0.02

H 0.03 0.02

K 0.03 0.02

R 0.03 0.01

The bacterial cells were incubated at 30 °C with shaking at 250 rpm, and optical density (OD600) was monitored.

Growth analysis of canonical variants of ECB at Tyr 72. Site directed mutagenesis was used to substitute Tyr

72 of pKTECB. The primers MK.20–39 and MK.40–59 were used for mutagenesis to the glycine (GGC), alanine

(GCG), serine (AGC), threonine (ACC), cysteine (TGC), valine (GTG), leucine (CTG), isoleucine (ATT), methi-

onine (ATG), proline (CCG), phenylalanine (TTT), tyrosine (TAT), tryptophan (TGG), aspartic acid (GAT), glu-

tamic acid (GAA), asparagine (AAC), glutamine (CAG), histidine (CAT), lysine (AAA) and arginine (CGT), re-

spectively. Individual KA12/pKIMP-UAUC clones harboring variant pKTECB plasmids containing mutations at

position 72 were isolated (termed pKTECB-Y72X20), and then washed, inoculated and cultured individually in

M9c medium supplemented with chloramphenicol (30 μg/ml), kanamycin (50 μg/ml) and tetracycline (100

ng/ml) at 30 °C with shaking at 250 rpm. Growth was monitored for 10 days (Table S1). For culture on solid me-

dia, a dilution series of the transformants (approximately 106, 105, 104, 103 and 102 cells) was spotted onto the

M9c or non-selective control M9c.FY supplemented with chloramphenicol (30 μg/ml), kanamycin (50 μg/ml) and

tetracycline (100 ng/ml). The plates were incubated at 30 °C for 10 days.

Reversion assay. Ec.ΔCM.BzF/pKTECB was grown in non-selective medium and harvested in late exponential

phase. Cells were washed three times with M9c, then three replicates of serial dilutions (approximately 1011, 1010,

109, 108 and 107 cells) were plated on M9c media containing chloramphenicol (30 μg/ml), kanamycin (50 μg/ml),

spectinomycin (50 μg/ml), tetracycline (100 ng/ml) and IPTG (1 mM). Growth was monitored at 30 °C for 10

days. The escape frequency was calculated as the escapees (c.f.u.) per total cells plated.

S5

Figure S4. Determination of quaternary structure of Ni-NTA column purified wild type mono functional EcCM (WT) and ECB by size-

exclusion column chromatography. (A) Overlaid FPLC spectra of WT (solid line) and ECB (dashed line). (B) The logarithm of the molec-

ular weight (Mr) of standard proteins was plotted against the Ve/Vo value (Ve, elution volume; Vo, void volume). The WT dimer (gray

closed circle) and ECB dimer (open circle) were located in the predicted range based on the elution volume.

Expression and purification of wild type E. coli chorismate mutase and ECB. To express wild type choris-

mate mutase, pET-CM plasmid was transformed into BL21(DE3) cells. Cells were inoculated into 10 ml LB sup-

plemented with ampicillin (100 μg/ml) and grown overnight at 37 °C. The overnight culture was added to 1,000

ml LB supplemented with ampicillin (100 μg/ml). When the OD600 reached 0.6, the culture flask was moved to 20

°C and incubated for additional 30 min, then induced with IPTG (0.5 mM). The culture was grown at 20 °C for

16 hours with shaking at 200 rpm.

The expression plasmid for ECB was constructed by ligating the NdeI and XhoI fragment of pKTECB

with the NdeI and XhoI fragment of pET-22b(+) to give pET-ECB. To express ECB, pET-ECB and pUltra-BzF

were co-transformed into the BL21(DE3) cells. Cells were inoculated into 10 ml LB supplemented with ampicil-

lin (100 μg/ml) and spectinomycin (50 μg/ml) and grown overnight at 37 °C. The overnight culture was added to

1,000 ml LB supplemented with pBzF (1 mM), ampicillin (100 μg/ml) and spectinomycin (50 μg/ml). When the

OD600 reached 0.6, the culture flask was moved to 20 °C and incubated for additional 30 min, then induced with

IPTG (0.5 mM). The culture was grown at 20 °C for 20 hours with shaking at 200 rpm.

To purify enzyme, cells were harvested by centrifugation at 6,000 g and stored at -78 °C. 15 g of thawed

cell pellet was suspended in 100 ml of buffer A (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole).

After sonication (10 min, output control 2.5, 50% duty cycle; Branson sonifier 450, Emerson, Ferguson, MO), the

cell lysate was centrifuged at 14,000g for 30 min at 4 °C, and supernatant was mixed with Ni-NTA agarose (Qi-

agen, Valencia, CA) and incubated for 1 hour at 4 °C. After washing with five column volumes of buffer A and

one column volume of buffer B (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 70 mM imidazole), the protein was

eluted with buffer C (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 250 mM imidazole). The buffer was exchanged

to DPBS (pH 7.4) by PD-10 column (Qiagen), then concentrated to approximately 5 mg/ml by Amicon Ultra 3

kDa mwco centrifugal filter (EMD Millipore). The proteins were analyzed by SDS-PAGE gels and mass spec-

trometry.

Size-exclusion column chromatography. The enzyme was purified and the quaternary structure was analyzed by

FPLC (ÄKTA purifier, GE Healthcare Life Sciences, Pittsburgh, PA) with Superdex 200 increase 10/300 GL

FPLC column (GE Healthcare Life Sciences) and DPBS (pH 7.4) as a running buffer. For the calibration curve,

gel filtration standards (Bio-Rad, Hercules, CA) were used (Figure S4).

S6

Figure S5. Characterization of purified wild type EcCM (WT) and ECB. (A) SDS-PAGE of WT and ECB. Q-TOF MS spectra and decon-

voluted mass spectra of WT (B) and ECB (C). (D) Comparison between calculated and observed mass (amu, atomic mass unit).

Mass spectrometry. High-resolution mass spectrometry was carried out on an Agilent 6520 accurate-mass quad-

rupole time-of-flight (Q-TOF) LC/MS instrument. Protein mass deconvolution from electrospray ionization (ESI)

mass spectrometry data was conducted by using Agilent Qualitative Analysis software (Agilent Technologies

Inc.) (Figure S5).

S7

Figure S6. Catalytic activity of wild type and ECB. Catalytic efficiency (kcat/Km) was calculated based on Michaelis-Menten kinetics for

the conversion of the chorismate to prephenate. SEC-purified homogeneous WT (closed circle) and ECB (open circle) were used. Error

bars represent standard deviation of triplicates.

Kinetic measurements. Catalytic efficiency (kcat/Km) was calculated based on spectroscopic measurement of the

conversion of the chorismate to prephenate at 274 nm (ε274 = 2,630 M-1cm-1) by 100 nM enzyme.9 Kinetic meas-

urements were performed at 25 °C in DPBS (pH 7.4), and bovine serum albumin (0.1 mg/mL) was used as an

additive. Initial rates were plotted over the range of chorismic acid concentration (0 μM, 31 μM, 63 μM, 125 μM,

250 μM, 500 μM, 1000 μM, 2000 μM), and the kinetic parameters were calculated using Michaelis-Menten kinet-

ics in GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). All experiments were conducted in triplicates

(Figures S2B and S6).

Figure S7. Circular dichroism experiments. (A) The mean residue ellipticity of wild type EcCM (WT) and ECB from 200 nm to 260 nm.

(B) Temperature-dependent unfolding curves for WT and ECB at 222 nm. WT: closed circle, ECB: open circle.

Circular dichroism spectroscopy. All circular dichroism (CD) experiments were performed on CD spectroph-

ometer model 420SF (AVIV, Lakewood, NJ), equipped with a Peltier temperature controller. CD spectra were

recorded with a 1 μM protein concentration in DPBS (pH 7.4) and a path length of 0.5 cm at 20 °C. Wavelength

scans from 260 to 200 nm were performed in 1 nm steps with signal averaging time of 7.5 s and a bandwidth of 1

nm. Temperature scanning experiments were performed at 222 nm with a bandwidth of 1 nm. The temperature

was raised from 20 °C to 95 °C in 0.5 °C steps with an equilibration time of 9 s and an averaging time of 7.5 s.

The midpoint of the unfolding transition, Tm, was obtained by calculating the maximum value of the first deriva-

tive with respect to temperature (Figure S7).

S8

Figure S8. The x-ray crystal structure of dimeric ECB is presented in orange and gray color. Electron density as a 2Fo – Fc contour map

(σ) is shown in meshed surface around pBzF (marine), surrounding residues (gray) and waters (magenta). Two active sites are illustrated as

red dashed circles.

Determination of the ECB x-ray crystallographic structure. The initial crystals grew under the previously

published conditions.10 The crystallization conditions were further optimized using vapor diffusion method in

both sitting and hanging drop manner in a 1:1 ratio of 1 µL of reservoir solution and 1 µL of protein. Protein solu-

tion consisted of 5 mg/mL of ECB in 10 mM Tris pH 7.8, 100 mM NaCl and 10% glycerol. Single crystals ap-

peared after 24 h and reached their maximum size within four days. The best crystals grew in 100mM Bicine pH

9.0, 100mM Na-Acetate and 32% PEG 8K at 22 °C. Selected crystals were harvested and frozen in liquid nitro-

gen with no additional cryo protectant for data collection.

Several data sets were collected at both Synchrotron Radiation Light Source (SSRL) beamlines 9-2, 12-2

and Advanced Photon Source (APS) beamlines 23-ID-D and 23-ID-B. The best X-ray diffraction data were col-

lected at APS using beamline 23-ID-B equipped with an Eiger-16M detector. Collected data were indexed and

integrated with XDS11 and scaled using Scala, a part of the CCP4 suite.12,13 Initial phase information was obtained

by molecular replacement using PHASER14 with the previously solved structure of ECB as a search model (PDB

ID code 1ECM). Waters were added using ArpWarp15 during the initial round of the refinement and the structure

was improved by iterative rounds of model building and refinement with the programs Coot and Refmac5.16,17 The crystals belong to space group P212121, with two molecules in the asymmetric unit (Figure S8). Crystallo-

graphic details and statistics are listed in Table S2.

S9

Table S2. Statistical parameters of crystallographic data, collection and refinement of ECB

Data collectiona

Wavelength (Å) 1.0332

Space group P212121

Unit cell dimensions [a, b, c (Å)] a = 44.86

b = 61.27

c = 62.85

Resolution range (Å) 23.75–2.00

Highest resolution shell (Å) 2.11–2.00

No. of observed reflections 108280

No. of unique reflections 12193

Multiplicity 8.9 (9.2)

Completeness (%) 99.7 (99.5)

<I/σI> 8.3 (2.1)

Rmerge (%) 18.3 (123.8)

Rpim (%) 6.5 (43.1)

CC1/2 (%) 99.2 (73.0)

Wilson B-factor 31.4

Refinement

Rwork (%) 21.4

Rfree (%) 24.0

No. atoms

Macromolecules 1536

Water 73

B-factor (Å2)

Macromolecules A: 41.8

B: 48.2

Solvent 48.1

R.m.s. deviations

Bond lengths (Å) 0.01

Bond angles (deg) 1.45

Ramachandran statistics (%)

Favored 100

Outliers 0

Molprobity score 0.96

PDB ID 5VHT a

Values in parentheses are for the highest-resolution shell

S10

Table S3. Primers used in the experiments

Name Sequence (5’ to 3’)

MK.01 GGG AAT TCC ATA TGA CAT CGG AAA ACC CGT TAC TGG CGC TG

MK.02 CCG CTC GAG CAA AGC CTG CTG AGT TAA TAC GGA ATC TTC

MK.03 CGG GGT ACC TCA GTG GTG GTG GTG GTG GTG CTC GAG C

MK.04 ACA TGC ATG CTT AAG ACC CAC TTT CAC ATT TAA GTT G

MK.05 GAA GAT CTC TTT TCT CTA TCA CTG ATA GGG AGT G

MK.06 TCC GCA AAT GAT CAA TTC AAG GCC GAA TAA GA

MK.07 GAT CAT TTG CGG ATT AGA AAA ACA ACT TAA ATG

MK.08 CGC GGA TCC TTA AGA CCC ACT TTC ACA TTT AAG TTG

MK.09 GGG AAT TCC ATA TGT ATA TCT CCT TCT TAA AGT TAA ACA AAA TTA TTT C

MK.10 CCC ATG CGA TTA CTC GCC TGT TCC AGC TCA TCA TTG AAG ATT CC

MK.11 GTA ATC GCA TGG GCG TCC AGA TGG TGC GCT TTA C

MK.12 CCC ATT AGA TTA CTC GCC TGT TCC AGC TCA TCA TTG AAG ATT CC

MK.13 GTA ATC TAA TGG GCG TCC AGA TGG TGC GCT TTA C

MK.14 GAT TTT AGA GCC AAT TAG AAA GAG ATT ATA AGT CGA CGC GTT TAA ACG GTC TCC AGC TTG

MK.15 CTC TTT ATC ATT TCA AAT TCG TCC ATA GAT CTG CAC CTC CTT TGT GAA ATT GTT ATC CG

MK.16 AGA TCT ATG GAC GAA TTT GAA ATG ATA AAG AGA AAC ACA TCT GAA ATT ATC AGC

MK.17 CAA GCT GGA GAC CGT TTA AAC GCG TCG ACT TAT AAT CTC TTT CTA ATT GGC TCT AAA ATC

MK.18

GGG AAT TCC ATA TGA CAT CGG AAA ACC CGT TAC TGG CGC TGC GAG AGA AAA TCA GCG CGC TGG ATG AAA AAT

TAT TAG CGT TAN NKG CAG AAC GGN NKG AAC TGG CCG TCG AGG TGG GAA AAG CCA AAC TGC TCT CGC ATC GCC

CGG TAC GTG ATA TTG ATC GTG AAC GCG ATT TGC TGG AAA GAT TAA TTA CGC TCG G

MK.19

CGG GGT ACC TCA GTG GTG GTG GTG GTG GTG CTC GAG CAA AGC CTG CTG AGT TAA TAC GGA MNN TTC AAT MNN

GAG CTG GAA MNN GCG AGT AAT CTA ATG GGC GTC CAG ATG GTG CGC TTT ACC GAG CGT AAT TAA TCT TTC CAG

CAA ATC GCG TTC ACG ATC AAT ATC ACG TAC CGG GCG ATG CGA GAG CAG TTT GGC TT

MK.20 CCA TGG CAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.21 CCA TGC GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.22 CCA TAG CAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.23 CCA TAC CAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.24 CCA TTG CAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.25 CCA TGT GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.26 CCA TCT GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.27 CCA TAT TAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.28 CCA TAT GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.29 CCA TCC GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.30 CCA TTT TAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.31 CCA TTA TAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.32 CCA TTG GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.33 CCA TGA TAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.34 CCA TGA AAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.35 CCA TAA CAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.36 CCA TCA GAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.37 CCA TCA TAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.38 CCA TAA AAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.39 CCA TCG TAT TAC TCG CAC GTT CCA GCT CGG TAT TGA ATA TTC C

MK.40 GAG TAA TGC CAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.41 GAG TAA TCG CAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.42 GAG TAA TGC TAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.43 GAG TAA TGG TAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.44 GAG TAA TGC AAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.45 GAG TAA TCA CAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.46 GAG TAA TCA GAT GGG CGT CCA GAT GGT GCG CTT TAC

S11

MK.47 GAG TAA TAA TAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.48 GAG TAA TCA TAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.49 GAG TAA TCG GAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.50 GAG TAA TAA AAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.51 GAG TAA TAT AAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.52 GAG TAA TCC AAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.53 GAG TAA TAT CAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.54 GAG TAA TTT CAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.55 GAG TAA TGT TAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.56 GAG TAA TCT GAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.57 GAG TAA TAT GAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.58 GAG TAA TTT TAT GGG CGT CCA GAT GGT GCG CTT TAC

MK.59 GAG TAA TAC GAT GGG CGT CCA GAT GGT GCG CTT TAC

S12

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