Yeast Display Nanobody

Library recovery and expansion
1. Place frozen aliquots of yeast nanobody library (NbLib) at 30 °C to thaw, such
that recoverable yeast exceed library diversity at least five-fold. For the full
naïve library, a total of 2.5×109 viable cells are required.

2. Recover yeast in 1 L of ‘Yglc4.5 –Trp’, shaking at 230 RPM, 30 °C, overnight. You
may want to take a small sample of the library cells immediately after
resuspension and plate on YPD agar to measure the diversity.

3. Expand to 3 L of medium and allow yeast to grow to stationary phase.
Approximately 48 hours, OD is usually between 10 and 30.

4. Measure OD600 of yeast to calculate density (OD600 of 1 ≈ 1.5 × 107 yeast)

5. Spin down cultures at 3500×g for 5 minutes and resuspend in ‘Yglc4.5 –Trp’
supplemented with 10% DMSO, such that final density is 1010 cells per mL.
Aliquot to 2 mL cryovials and freeze in a cell freezing chamber at -80°C
(Thermofisher Scientific cat# 5100-0001). Each vial should contain enough
viable cells upon recovery to reconstitute a full library.

Induction of nanobody expression

Nanobody expression is under the control of the GAL1 promoter, so nanobodies are
only produced on the cell surface when the yeast are grown in galactose-containing
medium.

1. Induce nanobody expression of NbLib by dilution of ≥10 x library diversity into –
Trp +galactose medium followed by shaking for 48 hours, 25 °C, 220 rpm. Use at
least 5 × 109 yeast cells for the inoculum to ensure no clones are lost in
passaging.

(We find best expression levels to be between 48 and 72 hours.)
Note about sterility: It is essential to maintain a sterile environment throughout your
selection. Unlike standard yeast work, your yeast cells will be passaged multiple times
over the course of weeks, increasing the possibility of contamination. We recommend
sterile filter tips and freshly sterilized media for every selection. Wipe down your
hands, bench, and pipettes with ethanol often during the selection process. A sterile
hood could be used as well, but usually a regular lab bench is fine so long as reasonable
care is taken.

Nanobody expression test

Before any selection it’s a good idea to confirm that nanobody expression was
effectively induced. We check this with a quick analytical flow cytometry assay as
described below.

1. Measure cell density of induced NbLib yeast (1 OD600 ≈ 1.5 x 107 cells/mL)
2. Add 500 μl of ‘selection buffer’ to two microcentrifuge tubes
3. Pipette approximately 1×106 induced yeast into each microcentrifuge tube and
centrifuge for 1 min at 3500 × g, 4 °C
4. Aspirate supernatant and resuspend pellet in 100 μl selection buffer
5. To one tube add ~0.5 μg of anti-HA antibody labeled with AlexaFluor647 or
AlexaFluor488 (to check for the percentage of cells expressing nanobody) and 1
μM final concentration of your protein labeled with a spectroscopically distinct
dye (to check if your antigen binds nonspecifically to yeast). Leave the other
tube unstained as a control
6. Rock both tubes at 4 °C for 15 minutes
7. Spin down cells for 1 minute at 3500 × g, 4 °C
8. Aspirate supernatant and resuspend cells in 500 μL of selection buffer
9. Spin down cells as before and aspirate supernatant
10. Resuspend cells in 100 μl of selection buffer and assess nanobody expression
level on flow cytometer using unstained sample to set gates

Note: The maximum number of expressing cells from the naïve library is ~25%. The
typical number of expressing cells is ~15-20%. If expression is on the low end (~8-12%)
consider increasing the number of cells used for nanobody selection to compensate.
Example flow cytometer yeast gating:

Nanobody Selection
The selection process below is at the heart of identifying clones that bind to your target
protein. This protocol describes how to separate antigen binding nanobody clones from
non-binders using magnetic cell sorting in two major steps: a “pre-clear” or negative
selection step, followed by a positive selection step. These are usually done in series on
the same day. The pre-clear step serves to deplete the library of any nanobodies that
bind to things like secondary antibodies, magnetic beads, etc. The positive selection
step isolates clones that bind to your antigen. After selection, you can expand your
enriched cells and repeat the process until binders are highly enriched.
11. Spin down a suitable number of cells to have at least 10-fold over library
diversity. For the naïve library (NbLib) you will need 5 × 109 cells. After one or
more rounds of selection a smaller number of cells may be used based on
estimated diversity. We recommend always using at least 1 × 107 cells, since
smaller numbers are difficult to manipulate. Perform centrifugation for 3
minutes at 3500 × g, 4 °C
12. Aspirate media and resuspend yeast via pipetting in 10 mL of chilled selection
buffer
13. Spin down yeast for 3 minutes at 3500 × g, 4 °C and aspirate supernatant
14. Resuspend yeast in 4.5 mL of selection buffer
15. Begin preclear to remove off-target binders from the naïve library by adding 500
μl of the ‘Miltenyi anti-fluorophore beads’ to the resuspended yeast (If using a
secondary for selection, add that to the preclearing reaction as well. ~200 nM is
typically sufficient to remove secondary binders)
16. Rock/Rotate slowly at 4 °C for 40 minutes
17. Approximately 20 minutes prior to the conclusion of incubation, place an ‘LD
column’ on the Miltenyi MACS magnet. Put a 15 mL sterile falcon under the
column and place a 50mL falcon cap over the top of the column to maintain
sterility. Add 5 mL of selection buffer to the LD column to allow it to equilibrate
before the next step. It will take ~20 minutes to flow through completely
18. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate buffer and
unbound beads
19. Resuspend by pipetting in 5 mL of selection buffer.
20. Flow yeast over equilibrated LD column, collect flowthrough in new sterile 15mL
centrifuge tube
21. Flush out remaining cells by washing column with an additional 2 mL of
selection buffer
22. Remove LD column from magnet and discard
23. Spin down flow through at 3500 × g at 4 °C and aspirate supernatant
24. To resuspended yeast, add 5mL selection buffer containing fluorophore labeled
antigen at a 1-1.5 μM concentration (if using a secondary to stain – preincubate
the secondary with antigen before staining yeast and use a molar concentration
of 2:3 secondary to antigen)
25. Rock or rotate slowly at 4 °C for 1 hour
26. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate supernatant
27. Resuspend yeast in 4.5 mL of selection buffer and 500 μl of anti-fluorophore
beads
28. Rock or rotate slowly at 4 °C for 20 minutes
29. Approximately 5 minutes before end of incubation, place an ‘LS column’ on the
Miltenyi MACS magnet. Put a 15 mL sterile falcon tube under the column and
place a 50mL cap over the top of the column to maintain sterility. Keep plunger
wrapped in package for later use. Add 5 mL of selection buffer to the LS column
and allow it to equilibrate before the next step. It will take ~5 minutes to flow
through completely
30. Spin down yeast for 3 minutes at 3500 × g at 4 °C and aspirate buffer and
unbound beads
31. Resuspend yeast by pipetting in 3 mL of selection buffer and spin down as above
32. Aspirate supernatant and resuspend yeast in 3 mL of selection buffer
33. Remove 50 μl “pre-LS” aliquot of resuspended yeast and set aside for later
analysis. Keep aliquot on ice.
34. Flow yeast over the equilibrated LS column and collect flow through in a new
sterile 15 mL centrifuge tube
35. Wash column with 8 mL of selection buffer 
36. Record approx. volume of flow through and remove 50 μl “flow through” aliquot
for later analysis.
37. Remove LS column from magnet and place over fresh sterile 15 mL centrifuge
tube
38. Add 5 mL of selection buffer to column and immediately use plunger to elute
cells
39. Remove 50 μl “elution” aliquot from eluted cells and set aside for later analysis.
Keep aliquot on ice.
40. Spin down elution at 3500 × g for 3 min at 4 °C and aspirate buffer taking care
not to disturb the pellet
41. Resuspend yeast in 3 mL of –TRP +glucose in a 14 mL Falcon culture tube
42. Shake at 30 °C for about 24 hours to recover.
43. Use flow cytometry to compare the number of yeast from pre-LS and elution
aliquots and use this ratio to get an estimate of the reduction in diversity of the
library.
Next steps: After recovering yeast, you can reinduce in galactose medium for
another round of selection, and iterate until you isolate binders. It’s a good idea to
use a different fluorophore for each selection round. We typically perform two
MACS selections followed by a FACS sort to isolate binders.
For a FACS sort, you can prepare your yeast by staining with antigen exactly as
described above. The details of how to sort will depend on the specific project and
sorting equipment being used. Once your selected library has a high proportion of
active clones (usually 20% or more of the total cells), you can move on to screening.
For screening, plate sorted yeast on –TRP plates for single clones. Grow single
clones in a sterile deep-well 96-well plate, with one clone per well. Then induce
expression and stain clones to identify those with the highest activity. Finally, a
yeast miniprep or colony PCR can be used to isolate nanobody DNA for analysis and
recombinant expression. Note that not every clone with activity on yeast retains
activity in recombinant form. A large majority of clones will show activity in both
formats, but there are some exceptions.
Appendix
3.8 g of –Trp drop-out media supplement (US Biological)
Yglc4.5 –Trp (1 liter)
6.7 g Yeast Nitrogen Base
10.4 g Sodium Citrate
7.4 g Citric Acid Monohydrate
10 mL Pen-Strep (10,000 units/mL stock)
20 g glucose
Adjust pH to 4.5; Sterifilter (or sterifilter a stock of 20% glucose and add with antibiotic
after autoclaving).
3.8 g –Trp drop-out media supplement (US Biological)
–Trp (+glucose or +galactose - 1 liter)
6.7 g Yeast Nitrogen Base
10 mL Pen-Strep (10,000 units/mL stock)
20 g glucose or galactose (glucose for normal growth and galactose for induction of
nanobodies)
Adjust pH to 6; Sterifilter (or sterifilter a stock of 20% glucose or galactose and add with
antibiotic after autoclaving).
20 g Bacto Peptone
YPAD (1 liter)
20 g Glucose
10 g Yeast Extract
18 mg Adenine
10 mL Pen-Strep (10,000 units/mL stock)
20 mM HEPES pH 7.5
Selection buffer*
150 mM sodium chloride
0.1% (w/v) bovine serum albumin
5 mM maltose
Sterile filter before use.
*Many other buffers will work equally well, depending on your protein you may need to
modify this. For GPCRs for instance, we usually add 0.1% (w/v) lauryl maltose neopentyl
glycol detergent.
No pH adjustment needed. Sterifilter or autoclave as appropriate
MACS accessories catalog numbers
Anti-Cy5/Anti-Alexa Fluor 647 MicroBeads Miltenyi Biotec Cat# 130-091-395
Anti-FITC MicroBeads Miltenyi Biotec Cat# 130-048-701
LD Columns Miltenyi Biotec Cat# 130-042-901
LS Columns Miltenyi Biotec Cat# 130-042-401

pYDS649HM

Library vector sequence: Nb.BV025 in pYDS649HM 8584 bp. Selectable in E. coli with ampicillin and in yeast by Trp dropout. Sequence with Gal1 promoter primer (“AATATACCTCTATACTTTAACGTC”).

GGTACCCGACAGGTTATCAGCAACAACACAGTCATATCCATTCTCAATTAGCTCTACCA CAGTGTGTGAACCAATGTATCCAGCACCACCTGTAACCAAAACAATTTTAGAAGTACTT TCACTTTGTAACTGAGCTGTCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTG GATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACATATCC ATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTA TCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGC TATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGAC TCTCCTCCGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGC GCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAG GAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATGAACGAATCAAATTAACAA CCATAGGATGATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGA AGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAACTGCATAACCACTTTA ACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTAATAAAAGTATC AACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACCCCGGAT CGAATTCAACCCTCACTAAAGGGCGGCCGCC

Mating factor alpha leader

ATGAGATTCCCATCTATCTTCACCGCTG TTTTGTTCGCTGCTTCTTCTGCTTTGGCTGCTCCAGCTAACACCACCACCGAAGACGAA ACCGCTCAAATCCCAGCTGAAGCTGTTATCGACTACTCTGACTTGGAAGGTGACTTCGA CGCTGCTGCTTTGCCATTGTCTAACTCTACCAACAACGGTTTGTCTTCTACCAACACCAC CATCGCTTCTATCGCTGCTAAGGAAGAAGGTGTTCAATTGGACAAGAGAGAA

MRFPSIFTAVLFAASSALAAPANTTTEDETAQIPAEAVIDYSDLEGDFDAAALPLSNSTNNGLSSTNTTIASIAAKEEGVQLDKRE

GCTAGC GCA

ASA

Nanobody coding sequence

CAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTG CGCCTGAGCTGCGCGGCGAGCGGCTCTATTTCTCTGTACGCTGAAATGGGCTGGTATC GCCAGGCGCCGGGCAAAGAACGCGAACTTGTTGCCGGTATTACTCGTGGTACTACTAC CTATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAAC ACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCG CGGCTGACTACGTTCAGGGTAAATCTCGTGTTATCCCGCATTACTATTGGGGCCAGGG CACCCAGGTGACCGTGAGCAGC

QVQLQESGGGLVQAGGSLRLSCAASGSISLYAEMGWYRQAPGKERELVAGITRGTTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADYVQGKSRVIPHYYWGQGTQVTVSS

GGATCC

HA epitope tag

TACCCATACGATGTTCCAGATTACGCT

649 stalk sequence

CAA ACATCCTCACCCTCTACAGAAGCATCAGTATCAACCTCTAGTACCTCTTCCTCTGCTTCA CAATCATCTGACCCAACTACAACATCTTCGTCCAGTTCATCGTCTTCCCCATCTTCCCAA TCTGAGGAAATTTCATCATCACCGACGGTCTCAACTACACCATCAACTTCTTCATCATCT TCCTCAATGACTTCAACCACCACAACAAAGTCAATCTCAACTTCCACTACAAGTTCAGC TCCAGTTACAGATGTGACAGTTTCCTCATCGCCTAGTAAATCTACCTCTACTTCGACAA GTACAGAAACATCTAAAACACCTACTTCAATGACAGAGTATACATCTAGTACATCGATA ATTTCGACTCCAGTTAGTCACTCGCAGACAGGTTTGTCGGCTTCATCAAGTTCATCATC TACAACATCCGGTTCTTCGTCCACTAAATCAGAAAGTTCGACAACATCTGGCTCTTCCC AGTCCGTGGAATCAACCTCCAGCCACGCCACTGTTCTTGCTAATTCCGCAGAAATGGTC ACAACATCCTCTAGTTCATCCTCAACATCCGAAATGTCATTAACTAGTACTGCTACCAG TGTACCAGTCTCATCTAGTAGCAGTACGACATATTCTACTAGCGCATCTACACAAGCCG TCACTACAACATCTTCTTCCACTGTATCTACAACTTCTTCTAGTACAACGTTAACAAGCG CATTCACACATTCTTCAACCACATCGTCCGACCAGCCACCCAGCGACACTACAAGTCCA TCTACGACACACGAACCTCATGTAACCACTCAGACGTCATCAGAAACATCTTCTTCTAA GTCATCTTCTACTTCTTCTTCAAGTACATCTCAAACCTCTGAGTCTGCAACACCATCCGA TTCCGTATCACCTGGAAGTTCTACATCAACATCTTCTAGTAGCACTTCTACTTCCACTTC TATTTCCAGTGGAGAAACGACAACTTCTTCTTCTTCATCATCTGCCACGACCACTTCTAA CAGCGCAACCTTGTCAGTCTCTACCACACAAACTTCGATTGAAGCCAGTTCATCTACTA CATCTACATCTAGTTCAACAATTACAACTTCAAGTAGTAGCGCTCACATATCGTCGAAA TCTCAATCTAGTATTACCTATCCCTCTTCCTCGACATCTTCATCTACATCGTCCTCAATTT CTAGCGAATCTGAAAGTTTTGAATCGACATCAGCAGAAGATGCTCCATCAACAGCACC TTCATCAAGTGTCTCTTCTAAGAGTTCTACCTCTACAACATCAAGCACATCGACATCTTC AAGCACTCCATCTCCATCACCATCTTCCGTGAGTTCTTCCTCCACCAGCTCATTGACAAC TTCTGCTGTATCAACACCAGCTACCTCTCATTCTCAAAGTACTGTAGTAACCACCACTA CTATTACTACATCAACAGGTCCAGTGATGTCTACGACAACAGCTTATTCTTCTAGTTCT ACTAGCAGCTCGGAATCTTCTGAGGTTCAGTCTGTCATGTCATCTACGCCTAGTTCAAC ATCAACAACAACCAGTTCGGAATCTACTTCATCTAGCTCCACAGCTTCTACCTCACCAT CAACCTCGCAAACTTTCGAAACTTCTCCTACTATAGGAGGTGTCCCCTCAACCACTTCA TTTGTCTCTACGCCAACAACGAAATTGTCGCACACTACTTCCACTATGACAGCACAGTC CGATAGTAAGTCTACCCACTCCTCAAGCACATCGACAGAAGATAAATCATCCACTGCTT CTGCAGTTGACGAAAGCACTACAACATCCACTTCCACGGAGTCTACTACATCAGTAACA TCAGGCACCTCCCATTCCGCTAAAGAATCTTCGTCAAATTCTAAGGTGTATAGTTCACA GACAGCACACTCATCCATAAGTGTTGCATCATCACCTAGTACAAAG

GGCGCC

CAAATC CAATCTTCTATGGTTGAAATCTCTACCTACGCTGGTTCTGCTAACTCTGTTAACGCTGG TGCTGGTGCTGGTGCTTTGTTCTTGTTGTTGTCTTTGGCTATCATC

TAATGATTAATTAA CTCGAGATCTGATAACAACAGTGTAGATGTAACAAAATCGACTTTGTTCCCACTGTACT TTTAGCTCGTACAAAATACAATATACTTTTCATTTCTCCGTAAACAACATGTTTTCCCAT GTAATATCCTTTTCTATTTTTCGTTCCGTTACCAACTTTACACATACTTTATATAGCTATT CACTTCTATACACTAAAAAACTAAGACAATTTTAATTTTGCTGCCTGCCATATTTCAATT TGTTATAAATTCCTATAATTTATCCTATTAGTAGCTAAAAAAAGATGAATGTGAATCGA ATCCTAAGAGAATTGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGC CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTG CAGCACATCCCCCCTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCC TTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCA TTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCC TAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGA CGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA ACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTA ACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG GTATTTCACACCGCAGGCAAGTGCACAAACAATACTTAAATAAATACTACTCAGTAATA ACCTATTTCTTAGCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTACACCATTT GTCTCCACACCTCCGCTTACATCAACACCAATAACGCCATTTAATCTAAGCGCATCACC AACATTTTCTGGCGTCAGTCCACCAGCTAACATAAAATGTAAGCTTTCGGGGCTCTCTT GCCTTCCAACCCAGTCAGAAATCGAGTTCCAATCCAAAAGTTCACCTGTCCCACCTGCT TCTGAATCAAACAAGGGAATAAACGAATGAGGTTTCTGTGAAGCTGCACTGAGTAGTA TGTTGCAGTCTTTTGGAAATACGAGTCTTTTAATAACTGGCAAACCGAGGAACTCTTGG TATTCTTGCCACGACTCATCTCCATGCAGTTGGACGATATCAATGCCGTAATCATTGAC CAGAGCCAAAACATCCTCCTTAGGTTGATTACGAAACACGCCAACCAAGTATTTCGGA GTGCCTGAACTATTTTTATATGCTTTTACAAGACTTGAAATTTTCCTTGCAATAACCGG GTCAATTGTTCTCTTTCTATTGGGCACACATATAATACCCAGCAAGTCAGCATCGGAAT CTAGAGCACATTCTGCGGCCTCTGTGCTCTGCAAGCCGCAAACTTTCACCAATGGACCA GAACTACCTGTGAAATTAATAACAGACATACTCCAAGCTGCCTTTGTGTGCTTAATCAC GTATACTCACGTGCTCAATAGTCACCAATGCCCTCCCTCTTGGCCCTCTCCTTTTCTTTT TTCGACCGAATTAATTCTTAATCGGCAAAAAAAGAAAAGCTCCGGATCAAGATTGTAC GTAAGGTGACAAGCTATTTTTCAATAAAGAATATCTTCCACTACTGCCATCTGGCGTCA TAACTGCAAAGTACACATATATTACGATGCTGTCTATTAAATGCTTCCTATATTATATA TATAGTAATGTCGTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTA AGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCC CGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTA TAGGTTAATGTCATGATAATAATGGTTTCTTAGGACGGATCGCTTGCCTGTAACTTACA CGCGCCTCGTATCTTTTAATGATGGAATAATTTGGGAATTTACTCTGTGTTTATTTATTT TTATGTTTTGTATTTGGATTTTAGAAAGTAAATAAAGAAGGTAGAAGAGTTACGGAAT GAAGAAAAAAAAATAAACAAAGGTTTAAAAAATTTCAACAAAAAGCGTACTTTACATA TATATTTATTAGACAAGAAAAGCAGATTAAATAGATATACATTCGATTAACGATAAGT AAAATGTAAAATCACAGGATTTTCGTGTGTGGTCTTCTACACAGACAAGATGAAACAA TTCGGCATTAATACCTGAGAGCAGGAAGAGCAAGATAAAAGGTAGTATTTGTTGGCGA TCCCCCTAGAGTCTTTTACATCTTCGGAAAACAAAAACTATTTTTTCTTTAATTTCTTTT TTTACTTTCTATTTTTAATTTATATATTTATATTAAAAAATTTAAATTATAATTATTTTTA TAGCACGTGATGAAAAGGACCCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCT GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCT GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCA TAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAA GGAGCTAACCGCTTTTTTTCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACA CGACGGGCAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGAC TCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGGAACGCCTGGTATC TTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCG TCAGGGGGGCCGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCG GGCAGTGAGCGCAACGCAATTAATGTGAGTTACCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCCTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGGAATTAACCCTCACTAAAGGG AACAAAAGCTG