lajeunesse mqp 4-25dr

MQP$BIO$JBD$0021------

Structure/Function,Characterization,of,Kekkon5,---

A-Major-Qualifying-Project-Report--

Submitted-to-the-Faculty-of--

WORCESTER-POLYTECHNIC-INSTITUTE--

In-partial-fulfillment-of-the-requirements-for-the--

Degree-of-Bachelor-of-Science--In--

Biology-and-Biotechnology--By--

___________________________________-Chloe-LaJeunesse-

-April-27,-2017-

----APPROVED:--________________________-Joseph-B.-Duffy,-Ph.-D.-Biology-and-Biotechnology-WPI-Project-Advisor-

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ABSTRACT,,Kekkon5- is- a- transmembrane- protein- found- in- Drosophila- melanogaster- with- an-

extracellular- region-comprised-of- leucine-rich-repeats- (LRR)-and-an- immunoglobulin- (Ig)-

domain- and- an- intracellular- region- containing- conserved- sequences-whose- functions- are-

not- well$understood.- To- further- investigate- the- significance- and- function- of- these-

intracellular-Short-Linear-Motifs-(SLiMs),-their-conservation-was-assayed-in-species-across-

the-phylum-Arthropoda- and-variants- in-which- the- second-and- third-motifs-were-missing-

from-Kekkon5-were-generated-and-assayed-in#vivo.!

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ACKNOWLEDGEMENTS,,,,

I-would- first- like- to- thank-Duff- for-making-this-project-possible.-You-have- inspired-

me-so-much-and-have-been-a-wonderful-advisor-from-the-day-you-accepted-me-into-your-lab-

my-freshman-year.-I-will-treasure-and-carry-all-of-the-advice-and-guidance-you-have-given-

me-into-my-future-as-a-scientist.-Thank-you-for-believing-in-me.-I-would-also-like-to-thank-

Alex-Putnam-for-all-of-your-help-in-the-lab-every-day-and-for-staying-late-to-help-me-with-my-

Western-blot.-I-was-really-scared-it-wasn’t-going-to-work,-but-you-were-calm-about-it,-and-it-

ended-up-working!-Without-you,-my-days-in-lab-would-have-been-a-lot-less-fun.-Thank-you-

Natalie- Farny- for- being- a- wonderful- mentor- throughout- iGEM- and- my- undergraduate-

career-alike.--You,-too,-have-inspired-me-more-than-I-can-say-and-I-am-extremely-grateful-to-

have-had-you-as-a-guide.--And-finally,-thank-you-to-the-extraordinary-faculty-and-staff-of-the-

Department-of-Biology-and-Biotechnology.-I-have-learned-so-much-from-all-of-you-and-I-am-

truly-grateful-for-the-time-I-have-spent-here-with-you.---

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TABLE,OF,CONTENTS-,,,ABSTRACT-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2--ACKNOWLEDGMENTS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3--TABLE-OF-CONTENTS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4--INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5--MATERIALS-AND-METHODS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10--RESULTS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18--DISCUSSION-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -25--REFERENCES-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28--APPENDIX-A:-SDM-PRIMER-SEQUENCES-AND-LOCATIONS-. . . . . . . . . . . . . . . . . . . . 30--APPENDIX-B:-ONGOING-TRANSGENIC-F1-CROSSES-IN-DROSOPHILA . . . . . . . . . . . . . 31--APPENDIX-C:-GENE-CONSTRUCTION-CONSTRUCTS . . . . . . . . . . . . . . . . . . . . . . . . . 32---

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INTRODUCTION,-

LIG#Proteins##

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LIGs- are- family- of- single$pass- transmembrane- proteins- that- contain- a- relatively-

unique- combination- of- leucine$rich- repeats- (LRRs)- and- immunoglobulin- (Ig)- domains.-

LRRs- are- a- relatively- common-motif- existing- in-hundreds-of- proteins- from-animal,- plant,-

prokaryotic,-and-even-viral-species.-Functionally,-they-are-commonly-associated-with-innate-

immunity,- mRNA- transport,- apoptosis,- neuronal- development,- due- to- their- ability- to-

recognize- and- bind- proteins- and- other- ligands.- (Ko- &- Kim,- 2007;- Ng- et- al.,- 2010).-

Structurally,-LRRs-are-comprised-of-a-core-repetitive-pattern-of-~-nine-amino-acid-resides-

(L$x$x$L$x$L$x$x$N)- that- can- be- repeated- dozens- of- times- within- one- molecule.- When-

stacked- together,- LRRs- adopt- a- concave,- horseshoe$like- structure.- (Padmanabhan- et- al.,-

2009).-

Like- LRRs,- Ig- domains- are- also- a- highly- common- structure.- They- are- more-

characteristically-involved-in-protein-binding,-and-are-most-commonly-found-in-proteins-of-

the- immune-system-such-as-antibodies-and-T$cell- receptors,-but-have-also-been- found- in-

other-cell$surface-receptors,-matrix-proteins,-and-enzymes-(Bork-et-al.,-1994).-Structurally,-

Ig-domains-consist-of-several-folded-antiparallel-β$sheets-connected-by-hypervariable-loop-

structures- that- lend- immunoglobulin- domains- their- ability- to- bind- a- wide- range- of-

structures-(Berg-et-al.,-2002).--

Though-LRRs-and-Ig$like-domains-individually-are-relatively-ubiquitous-in-proteins-

throughout-many- kingdoms- of- life,- their- combination- is- quite- uncommon- and- restricted-

evolutionarily.-In-the-human-proteome,-only-35-proteins-combine-LRR-and-Ig$like-domains,-

compared-to-over-350-human-proteins-containing-LRRs-and-over-1100-proteins-containing-

Ig$like-domains.-In-Drosophila#melanogaster,-only-nine-LIGs-exist-(Evans,-2006).-While-LIG-

proteins- have- yet- to- be-widely- characterized,- past- research- has- determined- that- certain-

LIGs-act-in-signaling-pathways-and-neuronal-development-(MacLaren-et-al.,-2004;-Mandai-

et-al.,-2009).--

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The#Kekkon#Subfamily#and#Kekkon#5#

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Most- LIGs- present- in-Drosophila# melanogaster- are-members- of- the- Kekkon- (Kek)-

subfamily—a-diagram-of-which-can-be-seen-in-Figure-1.-The-Kek-subfamily-is-comprised-of-

six- structurally- related- proteins- (Kek1$Kek6)- that- contain- seven- LRRs- and- one- Ig$like-

domain- in- their-extracellular- region- (MacLaren-et-al.,-2004).-The-cytoplasmic-domains-of-

the- Kekkon- proteins- are- significantly- more- diverse,- containing- unique- but- sometimes-

overlapping-combinations-of-short-stretches-of-highly-conserved-amino-acid-sequences,-or-

short-linear-motifs-(SLiMs)-(MacLaren-et-al.,-2004;-Davey-et-al.,-2006).-There-are-no-direct-

vertebrate- orthologs- of- the- Kek- proteins,- but- the- AMIGO/Alivin- subfamily- of- LIGS-

discovered- in-vertebrates-discovered- in-2003-contain- seven-LRR-and-one- Ig$like-domain,-

much- like- the- Kekkon- subfamily- (Chen- et- al.,- 2006).-While- the- structures- of- the- Kekkon-

proteins-are-well$known,- the- functions-of-each-protein-are-not-as-well$understood.-While-

roles- in-cellular-adhesion-and-neuronal-development-have-been-proposed-for- the-Kekkon-

subfamily- in- general,- Kek1- is- the- only- member- of- the- Kekkon- subfamily- with- a- well$

characterized-function-to-date,-having-a-defined-role-in-inhibiting-Epidermal-Growth-Factor-

Receptor-(EGFR)-signaling-(MacLaren-et-al.,-2004).--

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-Figure,1:,The,Kekkon,subfamily,of,LIGs.,,

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Structurally,- Kekkon5- is- similar- to- its- other- five- relatives,- with- an- extracellular-

region-comprised-of-seven-LRRs-and-one-Ig$like-domain.-The-intracellular-region-of-Kek5-is-

made-up-of-six-SLiMs-that-appear-to-be-highly-conserved—IC1,-IC2,- IC3,- IC4,- IC5,-and-IC6-

(the- PDZ- binding- domain)- (MacLaren- et- al.,- 2004).- Functional- studies- of- Kek5,- have-

suggested-a-role-as-a-regulator-of-bone-morphogenetic-protein-(BMP)-signaling-(Evans-et-

al.,- 2009).- The- BMP- signaling- pathway- is- present- in- many- species- from- Drosophila- to-

humans-and-participates-in-the-development-and-patterning-of-many-tissues.-Misregulation-

of-this-pathway-can-result-in-the-development-of-kidney-disease,-hypertension,-cancer,-and-

other- diseases- (Little- &-Mullins,- 2006).- In-Drosophila,- BMP- signaling- plays- a- key- role- in-

dorsoventral-and-tissue-patterning-in-the-embryo-and-the-development-of-the-wing-in-the-

adult- fly.-Misexpression- of-Kek5- in- the- imaginal-wing- disc,- a- structure- in- the- larvae- that-

gives-rise-to-the-wing,-results-in-several-defects-associated-with-the-BMP-signaling-pathway,-

such-as-the-absence-of-crossveins-in-the-wing-and-the-formation-of-ectopic-scutellar-bristles-

(Evans,- 2006;- Evans- et- al.,- 2009;-Menon,- 2013).-An- example- of- these-phenotypes- can-be-

seen-in-Figure-2-below.-

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-Figure,2:,Missing,anterior,crossveins,in,the,wing,(top),and,ectopic,scutellar,bristle,formation,

(bottom),upon,Kek5,misexpression,(Menon,,2013).,-

Further- studies- have- shown- that- the-misexpression- of- Kek5- in- the- imaginal-wing-

disc-produces-a-lethal-phenotype-in-which-cells-extrude-from-the-wing-imaginal-disc-and-die-

in- Caspase$dependent- manner.- Other- phenotypes- influenced- by- Kek5- misexpression-

(Figure- 3)- include- Armadillo- upregulation- at- the- adherens- junction- and- a- “large- cell”-

phenotype,-in-which-Kek5$misexpressing-cells-appear-to-be-much-larger-than-normal-cells-

(Menon,-2013).-

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Figure,3:,Panels,A,and,B,represent,Armadillo,staining,in,response,to,wildtype,Kek5,(A),or,Kek5%with,deleted,intracellular,domain,(B).,Armadillo,upregulation,is,seen,in,both,cases,while,the,“large,cell”,phenotype,is,seen,only,in,panel,A.,Panels,C,and,D,represent,light,microscope,images,showing,the,cell,extrusion,phenotype,in,response,to,wildtype,Kek5%(A),or,Kek5%with,deleted,

intracellular,domain,(D),(Menon,,2013).,-

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- While-the- importance-of-the-extracellular-region-of-Kek5-has-been-established,- the-

role-of-the-IC-domain-remains-largely-unclear.-In-order-to-better-understand-the-role-of-the-

cytoplasmic-tail,-variants-in-which-different-combinations-of-the-six-defined-IC-SLiMs-have-

been-deleted-have-been-created-and-expressed-in-Drosophila-(MacLaren-et-al.,-2004;-Evans,-

2006;-Evans-et-al.,-2009;-Menon,-2013).-Misexpression-studies-with- these-variants-reveal-

that-the-PDZ-binding-domain-(IC6)-is-necessary-for-Kek5-stabilization-and-localization,-and-

is-thus- indispensable-for-Kek5-function-(Evans,-2006;-Evans-et-al.,-2009).-With-respect-to-

the- remainder- of- the- intracellular- domain,- prior- work- indicates- it- is- critical- for- some-

processes,-but-not-others-(Evans-et-al.,-2009;-Figure-3).--While-evidence-points-towards-IC1-

being-dispensable-in-terms-of-overall-protein-function,-IC2,-IC3,-IC4,-and-IC5-all-appear-to-be-

significant.-In-particular,-the-second-and-third-SLiMs-of-Kek5,-IC2-and-IC3,-seem-to-affect-the-

formation- of- ectopic- scutellar- bristles,- the- cell- extrusion- and- death- phenotype,- and- the-

large$cell-phenotype-(Menon,-2013).-However,-because-only-deletion-variants-coupling-IC2-

and-IC3-with-each-other-and/or-other-SLiMs-have-been-made,-the-individual-effects-of-IC2-

and-IC3-on-Kek5-function-remain-unclear.--

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MATERIALS,AND,METHODS,#

CLUSTAL#Analysis#

Kek5- and- putative- Kek5- sequences- were- gathered- from-Drosophila# melanogaster-

(fruit- fly),- Anopheles# gambiae- (mosquito),- Pediculus# humanus# corporis- (body- louse),-

Acyrthosiphon# pisum- (pea- aphid),- Tribolium# castaneum# (flour- beetle),- Apis# mellifera-

(honeybee),- two- separate- instances- of- Acromyrmex# echinatior- (new- world- ants),- and-

Daphnia# pulex- (water- flea)- in- FASTA- format.- These- sequences- were- then- aligned- using-

EMBL$EBI’s-(Hinxton,-Cambridgeshire)-Clustal-Omega-software.-Amino-acid-residues-were-

color$coded- based- on- characteristics- such- as- hydrophobicity- and- polarity- for- easier-

recognition-of-shared-properties-between-residues.--

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Generation#of#DNA#Constructs#

The-pENTR$Kek5∆2- and- the-pENTRIKek5∆3- constructs-were- generated- synthesizing-

them-from-a#pENTRIKek5-(full$length)-template-through-site$directed-mutagenesis.-Figure-

4- shows- a- summary- of- this- process,- which- was- conducted- with- NEB’s- Q5- Site$Directed-

Mutagenesis-Kit.-In-two-separate-reactions-for-both-IC2-and-IC3,-8-μL-(approximately-25-ng)-

of-pENTR$Kek5-template-was-combined-with-Q5-Hot-Start-High$Fidelity-2X-Master-Mixed-

(12.5-μL),-distilled-water-(2-μL),-a- forward-primer,-and-a-reverse-primer-(10-μM,-1.25-μL-

each)-up- to-a- final-volume-of-25-μL.-These-primers- (Appendix-A)-were-designed- to- flank-

either- IC2- or- IC3- and- read- in- opposite- directions.- In- the- case- of- Kek5∆2,- only- IC2- was-

deleted.-In-the-case-of-Kek5∆3,#to-be-consistent-with-constructs-that-had-included-deletions-

of-IC3-generated-in-the-past,-the-deletion-spanned-from-the-beginning-of-IC3-to-just-before-

the-start-of-IC4.-The-PCR-reaction-for-both-of-these-constructs-was-run-for-25-cycles-with-an-

annealing-temperature-of-62°C-and-an-extension-time-of-2-minutes-and-45-seconds.-

After- the- PCR- was- complete,- a- kinase,- ligase,- and- DpnI- (KLD)- treatment- was-

prepared- for- each- reaction.-The-kinase- and- ligase- allow- the- ends-of- the-PCR-products- to-

ligate- together,- re$establishing- a- circular- vector.- DpnI- is- a- restriction- enzyme- that- only-

digests-methylated-DNA.-Because-the-original-pENTRIKek5#template-was-synthesized-in-E.#

coli,-the-DNA-has-been-methylated.-The-pENTR$Kek5∆2-and-the-pENTRIKek5∆3-constructs,-in-

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contrast,-were-synthesized-in-a-PCR-reaction-and-thus-have-not-been-methylated.-Because-

of- this,- the- addition- of-DpnI-will- degrade- the- template-DNA-while- preserving- the-newly$

synthesized- pENTR$Kek5∆2- or- pENTRIKek5∆3- constructs,- ensuring- that- the- resulting- DNA-

will-have-no-template-present.--

Once- the-KLD- treatment-was- complete,- the-DNA-was- transformed- into-DH5α-cells-

and-plated-onto-on-LB-agar+kanamycin-plates.-Colonies-were-picked-and-grown-up-in-5-mL-

of- liquid- culture- (LB+kanamycin)- and- miniprepped- using- QIAGEN’S- QIAPREP- Spin-

Miniprep-Kit.-Minipreps-of-selected-clones-were-analyzed-via-gel-electrophoresis-and-clones-

likely-to-be-positive-for-pENTR$Kek5∆2-or-pENTRIKek5∆3-were-sequenced-by-Eton-Bioscience-

Inc.- (Boston,- MA)- and- were- verified- in- lab- by- constructing- contigs- of- the- sequenced-

fragments-of-each-construct-in-Sequencher.--

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,Figure,4:,Site\directed,mutagenesis,in,the,generation,of,Kek5∆2,and,Kek5∆3,pENTR%constructs.-

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Once-pENTR$Kek5∆2-or-pENTRIKek5∆3-were-determined-to-be-successfully-generated,-

they- needed- to- be- transferred- to- an- expression- vector- by- making- use- of- the- Gateway-

cloning-system.-An-overview-of-this-process-can-be-seen-in-Figure-5.---

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-Figure,5:,LR/BP,reactions,in,the,Gateway,cloning,system,in,relation,to,Kek5,constructs.,

,

To- each- reaction- 1- μL- (approximately- 100- ng)- of-pENTR$Kek5∆2- or-pENTRIKek5∆3-

template,-1-μL-(150-ng)-of-pDEST-vector-(in-this-case,-pUASTaGFP),-6-μL-of-distilled-water,-

and-2-μL-of-Invitrogen-Gateway-LR-clonase-were-added.-The-LR-clonase-contains-integrase-

and-excisionase,-two-recombination-enzymes-from-the-lambda-bacteriophage-that-catalyze-

recombination-between-two-vectors-containing-attL-and-attR-sites.-The-attL-and-attR-sites-

become-attP-and-attB-sites,-respectively,-resulting-in-an-expression-clone-of-Kek5-deletion-

variants- flanked- by- attB- sites.- The- LR- reactions-were- left- to- incubate- at- 25°C- for- 1- hour-

before- 1- μL- (2- μg)- of- Proteinase-K-was- added- to- digest- the- recombination- enzymes- and-

terminate-the-reaction.--

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Once-the-reactions-were-complete,-they-were-again-transformed-into-DH5α-cells-and-

plated-onto-on-LB-agar+ampicillin-plates-to-select-for-the-expression-clone.-Colonies-were-

then- picked- and- grown- in-mL- of- liquid- culture- (LB+ampicillin),- then-miniprepped- using-

QIAGEN’S-QIAPREP-Spin-Miniprep-Kit.-Since-PCR-is-not-involved-in-this-cloning-step-there-is-

little-opportunity-for-replication-errors-or-mutation-in-the-LR-reaction-process.-Therefore,-

only-reactions-for-the-∆2-and-∆3-junctions-and-the-GFP-region-of-each-construct-were-sent-

out- for- sequencing- by- Eton- Bioscience- Inc.- (Boston,- MA).- Because- the- original- Kek5-

sequence- did- not- include- a- stop- codon,- when- translated,- the- Kek5- ∆2- and- ∆3- deletion-

variants-will-be-tagged-with-the-eGFP-sequence-included-in-the-original-pUASTaGFP-vector,-

which-does-include-a-stop-codon.-

Upon-verification-of-the-correct-Kek5∆2-or-Kek5∆3-inserts-in-pUASTaGFP,#midipreps-of-

each- constructs- were- prepared- with- QIAGEN’S- QIAGEN- Plasmid-Midi- Kit- to- ensure- that-

enough-DNA-would-be-generated-to-be-sent-off-for-injection-into-Drosophila#melanogaster-

embryos.,

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Gal4/UAS#System#

The-Gal4/UAS-system-is-a-gene-regulatory-system-used- in-research- for-conducting-

gain-of- function-studies- through-controlled-gene-expression-(Duffy,-2002).-The-Gal4-gene-

encodes- a- protein- capable- of- regulating- the- transcription- of- genes- associated- with- an-

Upstream- Activating- Sequences- (UAS)- by- directly- binding- to- the- UAS- to- activate-

transcription.-Originally-discovered-in-the-yeast-Saccharomyces#cerevisiae,- this-system-has-

been- adapted- for- use- in- many- organisms.- For- organismal- gain- of- function- studies- in-

Drosophila,# this- system- is- employed- through- the- crossing-of-driver- (Gal4)-and- responder-

(UAS)- lines.- Lines- of- transgenic- flies- called- responders- that- contain- the- gene- of- interest-

associated-with-UAS-elements-possess-this-gene,-but-do-not-express-it,-as-no-Gal4-is-present-

to- activate- transcription.-These- responders- are-mated- to- lines- of- flies- expressing-Gal4- in-

different- tissues-and-patterns,- the-drivers.-This-results- in-progeny-expressing- the-gene-of-

interest- in- the- tissues- associated- with- the- expression- of- Gal4- in- the- driver- lines- (Duffy,-

2002).- An- overview- of- this- system- is- featured- in- Figure- 6.- This- system- allows- for- the-

misexpression- of- genes- in-Drosophila# (and- other- eukaryotes)-while- also- allowing- for- the-

maintenance-of-a-potentially-lethal-or-otherwise-harmful-gene-in-a-line.--In-cell-culture,-the-

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Gal4/UAS- system- is- also- used- for- gain- of- function- studies- through- the- co$transfection- of-

driver-and-responder-sequences-directly- into- the-culture- (Klueg-et-al.-2002).-The-drivers-

used- in- these- cases- can- be- constitutively- expressed,- such- as- the- ArmIGal4# driver,- or-

inducible,- such- as- the- mtIGal4# driver,- for- which- expression- can- be- induced- by- the-

introduction-of-copper-or-other-heavy-metals-(Klueg,-et-al.,-2002).--

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#Figure,6:,The,Gal4,misexpression,system,in,Drosophila%melanogaster,(Duffy,,2002).-

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Generation#of#Transgenics#

50-μG-each-of-pUASTaKek5∆2GFP-and-pUASTaKek5∆3GFP#DNA-was-sent-to-Best-Gene,-

Inc.- (Chino- Hills,- CA)- for- injection- into- w1118- embryos.- After- injection,- the- larvae- were-

mailed- back- to-Duffy- lab- for- transgenic- screening.- Once- the- larvae- eclosed,- the- resulting-

flies-(generation-G0)-were-single$pair-mated-to-2$3-w1118-males-or-females.-The-pUASTaGFP-

vector- the-Kek5∆2- and-Kek5∆3- genes-were- transposed- into- contained- a- copy- of- the-white-

gene-under-its-own-set-of-regulatory-elements.-Therefore,-any-F1-progeny-from-the-G0-cross-

with- eye- color- are- likely- to- contain- the- transgene.- The- F1- offspring- of- the- G0- cross-were-

screened- for- eye- color,- and- any- putative- transgenics- gathered- from- separate- G0- crosses-

were- considered- independent- insertion- events.- Putative- transgenic- offspring- (generation-

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F1)- for- each- line- were- single$pair- mated- to- 2$3-w1118- males- or- females.- This- mating- of-

putative- transgenics- to-w1118- ensures- the- amplification- of- all- transgenic- lines- for- stable-

stock-generation-and-future-gene-mapping.-

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Transfection#in#Cell#Culture##

# To- confirm- Kek5∆2GFP- and- Kek5∆3GFP- expression- prior- to- the- generation- of- the-

transgenics,-pUASTaKek5∆2GFP-and-pUASTaKek5∆3GFP#were-transfected-into-S3-cells,-a-cell-

line-derived- from-male-Drosophila#melanogaster- embryonic- cells.- First,- each-DNA-sample-

was-purified-using-a-phenol:chloroform-extraction.-An-equal-volume-of-phenol:chloroform-

was- added- to- a- 20- μL- aliquot- of- each- sample- and- after- vortexting,- the- samples- were-

centrifuged-for-2-minutes-at-room-temperature.-To-each-tube,-5-M-NaCl-(in-a-ratio-of-1:20)-

and-100%-EtOH-(in-a-ratio-of-2:1)-were-added-to-precipitate-the-DNA.-The-solutions-were-

incubated-on-ice-for-10-minutes,-then-centrifuged-to-pellet-the-DNA.-Then,-the-DNA-pellet-

was-washed-with-70%-EtOH,-centrifuged-again,-decanted,-and-left-to-air-dry.-Once-dry,-the-

pellets-were-taken-up-in-TE-buffer.-The-concentration-of-each-sample-was-measured-on-a-

NanoDrop,-and-each-construct-was-diluted-to-a-concentration-of-100-ng/µL.-

- Prior- to- transfection,- S3- cells- were- split- into- a- T75- flask- maintained- at- normal-

growth-conditions-(25°C,-no-CO2).-One-day-before-transfection,-on-the-day-the-cells-would-

normally- be- split,- the- S3- cells- in- the- T75- were- resuspended- in- Schneider’s- Media- and-

transferred- to-a-15-mL-conical- tube.-An-aliquot-of- cells-was- taken- from-this- tube,-diluted-

1:10,-and-counted-on-a-hemocytometer.-Then,-according-to-the-appropriate-dilution-factor,-

5-x-106-cells-were-seeded-into-each-well-of-a-6-well-cell-culture-plate-at-a-final-volume-of-1.6-

mL-per-well.-The-plates-were- incubated-at-normal-growth-conditions-for-24-hours-until-a-

confluence-of-approximately-80$100%-was-reached.-

- The-next-day,-four-wells-were-co$transfected-with-400-ng-of-ArmIGal4-(a-constitutive-

driver)- and- 400- ng- of- either-pUASTaKek5wtGFP,# pUASTaKek5∆2GFP,-pUASTaKek5∆3GFP,- or-

nothing-(negative-control)-in-EC-buffer.-Once-Enhancer-and-Effectine-Transfection-reagent-

were-added-and-incubated-in-sequence,-the-media-of-each-well-was-aspirated-and-each-well-

was-washed-with-1X-PBS,-which-was- then-aspirated,-as-well.-1.6-mL-and-0.6-mL-of- fresh-

Schneider’s-Media-was-added-to-each-well-and-each-co$transfection-mix,-respectively.-Each-

16-

!

co$transfection-mix-was-added-to-an-appropriately-labelled-well-and-the-plates-were-left-to-

incubate-at-normal-growth-conditions-overnight.-

#

GFP#Imaging-

Because- the- Kek5# constructs- were- tagged- with- GFP,- the- wild- type- and- variant-

proteins-produced-should-be-visible-through-fluorescent-microscopy.-In-order-to-visualize-

the-Kek5$GFP-proteins-and-observe-expression-and-localization-patterns,-the-co$transfected-

plate-was-viewed-under-under-Zeiss-SteREO-Discovery.V12-microscope-at-100X-either-at-

480- nm- (GFP- fluorescence)- or- under- incandescent- light- (cell- images).- Images- were-

captured-with-a-monochrome-Zeiss-Axiocam-and-processed-with-the-Axiovision-software.--

-

Trypan#Blue#Vital#Stain##

At-day-6-post$transfection,-an-aliquot-of-100-μL-of-resuspended-cells-was-taken-from-

each-co$transfection-well-and-combined-with-100-μL-of-Trypan-blue,-which-is-a-stain-only-

taken- up- by- dead- cells.- For- each- co$transfection,- 10- μL- of- this- Trypan- blue-mixture-was-

loaded-into-a-hemocytometer-and-the-numbers-of-living-and-dead-cells-were-counted-in-two-

separate-squares-and-totaled.-The-percentage-of-dead-cells-to-the-total-number-of-cells-for-

each-co$transfection-was-then-calculated.-

#

Western#Blot#

In-order-to-detect-potential-Kek5wtGFP,-Kek5∆2GFP,-and-Kek5∆3GFP-expression,-125-

μL-(approximately-2-x-107-cells)-of-each-co$transfection-was-aliquoted,-spun-down,- taken-

up- in-1-mL-of- sample-buffer- (12-mM-TrisCl,-5%-glycerol,-0.4%-SDS,-2.9-mM-βME,-0.02%-

bromophenol-blue),-and-stored-at-$20°C.-Just-before-loading,-two-aliquots-of-5-μL-and-20-μL-

for- each- sample- were- made- and- lysed- by- boiling- for- 5- minutes.- Samples- were- run- on-

precast- 7.5%-polyacrylamide- gels- from-BIORAD-at- 20mA- for- 1- hour.- Proteins-were- then-

transferred-from-the-gel-to-a-nitrocellulose-membrane-100V-for-1-hour.-In-order-to-confirm-

the- transfer,- the- membrane- was- Ponceau- stained,- then- blocked- for- 1- hour.- Then,- the-

membrane-was-blocked-in-5%-NFDM-in-TBST-(100-mM-Tris,-150-mM-NaCl,-0.1%-Tween$

20)- for- 1- hour,- after- which- the- membrane- was- rinsed- and- incubated- with- the- primary-

antibody-(monoclonal-α$GFP)-diluted-in-0.5%-NFDM-in-TBST-overnight-at-4°C.--

17-

!

The-next-day,-the-membrane-was-washed-in-TBST-and-incubated-with-the-secondary-

antibody- (goat- α$mouse,- diluted- 1:20,000- in- 5%- NFDM- in- TBST)- for- one- hour- at- room-

temperature.- The- membrane- was- once- again- washed- in- TBST- and- incubated- in- HRP-

substrate-for-10-minutes,-then-photographed-with-chemiluminescent-imaging.,

-

% %

18-

!

RESULTS##

There- is-much- evidence- to- support- links- between- the- IC- domain- of- Kek5- and- its-

overall- function.- Phylogenetic- analysis- reveals- that- all- SLiMs- found- in- the- IC- domain- of-

Kek5-show-a-relatively-high-level-of-conservation-in-closely-related-species,-suggesting-an-

evolutionary-advantage-to-maintaining-these-sequences-(MacLaren-et-al.,-2004;-Talwar-and-

Duffy,- personal- communication).- Additionally,- past- research- has- shown- that- both- the- IC-

domain-as-a-whole-and-certain-combinations-of-IC-SLiMs-have-large-effects-on-the-function-

of-Kek5-(Evans,-2006;-Menon,-2013).-Among-these-combinations,-IC2-and-IC3-in-particular-

have- been- shown- to- have- a- significant- effect- on- phenotypes- associated- with- Kek5-

misexpression-when-deleted-alongside- IC1.-However,-variants- in-which- IC2-and- IC3-have-

been- individually- deleted- have- not- yet- been- successfully- generated.- Therefore,- a- further-

investigation- on- the- both- the- significance- of- these- domains- and- their- individual-

contributions-to-Kek5-function-was-undertaken.--

#

Phylogenetic#Analysis#of#IC2#and#IC3#Conservation#in#Kekkon5#Homologs-

An- alignment- of- Kek5- orthologs- and- putative- orthologs-was- created- using- Clustal-

Omega.-A-sampling-of-Kek5-protein-sequences- for-closely$related-and- less-closely$related-

members- of- Hexapoda- were- analyzed- as- well- as- one-member- of- Crustacea.- The- species-

sampled- for- this-alignment-were,- in-order-of- sequence- in-Figures-7-and-8,-Daphnia#pulex-

(water- flea),-Anopheles#gambiae- (mosquito),-Drosophila#melanogaster- (fruit- fly),-Pediculus#

humanus# corporis- (body- louse),- Acyrthosiphon# pisum- (pea- aphid),- Tribolium# castaneum#

(flour- beetle),- Apis# mellifera- (honeybee),- and- two- separate- instances- of- Acromyrmex#

echinatior-(new-world-ants).--

Figure- 7- shows- the-portion- of- the- alignment- featuring- the- SLiM- $- IC2- (fingerprint-

PDLL).- The- alignment- of- IC2- reveals- that- sequences- highly- similar- to- Drosophila#

melanogaster’s-Kek5-IC2-exist-in-all-sampled-sequences.-

-

-

19-

!

-Figure,7:,Clustal,Omega,alignments,of,Kek5,IC2,in,representative,species,of,Crustacea,(Dpulk5/6),,

and,Chelicerata,(MoKek5),,and,Hexapoda,(all,others).,-

Likewise,- Figure- 8- shows- the- portion- of- the- alignment- featuring- SLiM- $- IC3-

(fingerprint-TIPR).-This-alignment-found-that-sequences-closely-related-in-sequence-to-the-

Kek5-IC3-region-of-Drosophila#melanogaster-exist-in-all-of-the-aligned-sequences.-,

-

-Figure,8:,Clustal,Omega,alignments,of,Kek5,IC3,in,representative,species,of,Crustacea,(Dpulk5/6),,

and,Chelicerata,(MoKek5),,and,Hexapoda,(all,others).,,-

#

Design#of#Novel#Kek5∆2!and$Kek5∆3!Constructs#

Previously,-four-variants-of-Kek5-in-which-IC2-and-IC3-were-deleted-in-combination-

with-other-SLiMs-have-been-generated.-These-constructs-yielded-high-rates-of-lethality-and-

additional- phenotypic- effects- (i.e.- extrusion,- cell- death,- large- cell- phenotype)- when-

expressed- in- Drosophila# melanogaster- (Menon,- 2013).- To- form- a- better- idea- of- the-

individual-effects-and-contributions-of-IC2-and-IC3,-constructs-of-Kek5∆2-and-Kek5∆3-were-

first-synthesized-in-a-pENTR-vector-via-site$directed-mutagenesis-of-an-existing-full-length-

pENTRIKek5-construct.-Then,-Kek5∆2-and-Kek5∆3-were-shuttled-from-the-entry-vector-into-

20-

!

the- expression- vector- pUASTaGFP.- Figure- 9- shows- the- structure- of- the- Kek5- gene- for-

wildtype,-Kek5∆2,-and-Kek5∆3#with-their-respective-lengths.-

-

-Figure,9:,Wildtype,Kek5,and,novel,Kek5,variants:,Kek5∆2,and,Kek5∆3.,

#

Both-the-pENTR-and-pUASTaGFP-constructs-for-each-deletion-variant-were-sequence-

verified.-The-pENTR#constructs-for-Kek5∆2-and-Kek5∆3-were-fully-sequenced-from-the-start-

codon-through-the-end-of-the-gene-to-verify-that-no-mutations-had-accumulated-during-the-

polymerase- chain- reaction.-Because- there- is- little- risk- of-mutation-upon- shuttling- a- gene-

from-one-P$element- vector- to- another- via- the-Gateway- cloning- system,- only- the-deletion-

junction- and- terminal- region- of- pUASTaKek5∆2GFP- and- pUASTaKek5∆3GFP- needed- to- be-

sequenced-to-sufficiently-verify-the-final-constructs.-

-

Generation#of#Transgenic#Lines-

Larvae- injected- with- pUASTaKek5∆2GFP- and- pUASTaKek5∆3GFP# constructs- were-

received-after-mailing-DNA-off- for- injection.- In- total,-143-adult- flies-(Generation-G0)-were-

received- from- these- injections—48- injected-with-pUASTaKek5∆2GFP#and-95- injected-with-

pUASTaKek5∆3GFP.#Each-of-these-flies-were-single$pair-mated-to-2$3-W1118-males-or-females.-

From-these-crosses,-7-putative-Kek5∆2#and-9#putative#Kek5∆3#F1-transgenics-were-collected.-

These-putative-transgenics-have-been-crossed-to-2$3-W1118-males-or-females,-depending-on-

the-sex-of-the-transgenic.--

Figure-10-shows-a-summary-of-the-overall-plan-and-current-state-of-the-generation-

of-transgenic-Kek5∆2#and-Kek5∆3#lines.-These-lines-have-yet-to-be-mapped-and-stabilized,-but-

LRR Ig Tm 1 2 4 5 PDZ 3

LRR Ig Tm 1 4 5 PDZ 3

LRR Ig Tm 1 2 4 5 PDZ

Kek5 wt 2751 bp

Kek5∆2

2664 bp

Kek5∆3

2580 bp

21-

!

the- process- of- doing- so- for- each- line- has- already- begun.- After- the- lines- are-mapped- and-

stable-stocks-are-produced,-functional-tests-can-be-begin-by-crossing-the-Kek5∆2#and-Kek5∆3#

lines-to-lines-expressing-a-Gal4-driver.--

-Figure,10:,Flowchart,of,the,plan,and,progress,for,generating,and,testing,stable,stocks,of,Kek5∆2,and,

Kek5∆3,deletion,variants,in,Drosophila%melanogaster.%,--

-

Expression#and#Localization#of#Kek5#Variants#in#Cell#Culture#

- In- order- to- confirm- the- expression- and- localization- of- the- two- deletion- variants,-

pUASTaKek5∆2GFP-and-pUASTaKek5∆3GFP,-these-constructs-were-co$transfected-into-an-S3-

cell- line- with- a- constitutive- ArmIGal4- driver.- At- 2,- 5,- and- 6- days- post$transfection,-

fluorescent- and- bright- field- microscopy- images- were- taken- of- each- of- the- two- deletion-

variant- constructs- and- positive- (ArmIGal4- +- pUASaKek5wtGFP)- and- negative- (ArmIGal4-

alone)-transfection-controls.-These-images-have-been-compiled-into-Figure-11-below.--

Generate Constructs (2)

Functional Tests

Design Variants

Create Transgenics (48 Kek5∆2 crosses, 95 Kek5∆3 crosses)

Map & Create Stable Stocks (all lines)

Localization/Expression Studies (GFP-based)

Misexpression Studies (A9-Gal4 & Ptc-Gal4 by all lines)

22-

!

-

Figure,11:,Fluorescent,microscopy,of,Arm9Gal4,+,UAS9Kek5wtGFP,,UAS9Kek5∆2GFP,,and,UAS9Kek5∆3GFP,co\transfections,and,Arm9Gal4,control,on,day,2,,day,5,,and,day,6,after,transfection.,Light,microscope,

images,are,displayed,in,each,corner,inset.,-

23-

!

- On-day-2-post$transfection,-the-UASIKek5wtGFP,#UASIKek5∆2GFP,-and-UASIKek5∆3GFP-

ArmIGal4-co$transfections-all-display-GFP-that-appears-to-be-localized-to-the-membrane-of-

each-cell-consistent-with-the-localization-and-expression-pattern-of-Kek5.-As-expected,-the-

control,-which-has-been-transfected-with-only- the-ArmIGal4-driver,-does-not-display-GFP.-

On-day-5-post$transfection,-the-UASIKek5wtGFP-and-UASIKek5∆2GFP-transfections-display-a-

similar- pattern- with- an- apparent- increase- in- levels- and- proportion- of- cells.- However,-

surprisingly-starting-at-day-5-post$transfection,- the-UASIKek5∆2GFP- co$transfection-ceases-

to-display-a-GFP-signal.-This-same-pattern-is-seen-on-day-6-post$transfection.-

#

Cytotoxic#Effect#of#Kek5∆2-#

The- data- gathered- from- the- GFP- localization- and- expression- analysis- in- the-

transfections-revealed-a-decrease-in-GFP-expression-starting-at-day-5-post$transfection-for-

the-ArmIGal4-+-UASIKek5∆2GFP-co$transfection.-Based-on-this-data,-it-was-hypothesized-that-

the-cells-in-the-ArmIGal4-+-UASIKek5∆2GFP#co$transfection-were-dying.-In-order-to-test-this,-

a-vital-stain-(trypan-blue)-was-conducted-using-aliquots-of-cells- from-each-transfection-at-

day- 6- post$transfection.- - A- bar- graph- of- the- percentages- of- dead- cells- observed- for- each-

transfection-can-be-seen-in-Figure-12.--

-

,Figure,12:,Percentage,of,dead,cells,in,trypan,blue,cell,counts,of,Arm\Gal4,+,UAS\Kek5wtGFP,,UAS\Kek5∆2GFP,,and,UAS\Kek5∆3GFP,co\transfections,and,Arm\Gal4,control,at,day,6,after,transfection.,

0%

10%

20%

30%

40%

50%

60%

UAS Kek5wt GFP! UAS Kek5Δ2 GFP!

UAS Kek5Δ3 GFP!

Arm-Gal4!

% c

ell d

eath!

Effect of IC Variants on Cell Viability!

24-

!

The- counts- of- dead- versus- living- cells- in- each- co$transfection- aliquot- reveal- a-

percentage-of- cell-death- that- is-dramatically- increased- in- the-UASIKek5∆2GFP- transfection-

relative-to-the-other-three-transfections.-The-percentage-of-dead-cells-observed-in-the-UASI

Kek5wtGFP,-UASIKek5∆3GFP,-and-the-Arm$Gal4-transfections-is-consistently-close-to-or-below-

10%.- In- contrast,- the- percentage- of- dead- cells- seen- in- the- UASIKek5∆2GFP- transfection-

counts-is-nearly-50%,-suggesting-a-higher-rate-of-death-in-cells-expressing-Kek5∆2GFP.-

-

Analysis#of#Kek5#Variant#Protein#Expression-

In-another-attempt-to-confirm-the-expression-of-Kek5∆2GFP-and-Kek5∆3GFP,-an-anti$

GFP-western-blot-was-run-with-cell-lysates-at-day-6-post$transfection-from-each-of-the-four-

co$transfections.- Figure- 13- is- an- image- of- the- resulting- blot- with- lanes- labelled- and- the-

expected-sizes-Kek5wtGFP-and-UAS$Kek5∆3GFP-listed.--

,Figure,13:,Anti\GFP,Western,blots,of,Arm\Gal4,+,UAS\Kek5wtGFP,,UAS\Kek5∆2GFP,,and,UAS\Kek5∆3GFP,

co\transfections.,-

Western-blot-showed-that-Kek5wtGFP-(lane-1)- is-running-close-to- its-expected-size-

(130-kDa)-and-that-UAS$Kek5∆3GFP-(lane-3)-is-running-slightly-below-at-what-seems-to-be-

its-expected-size-(124-kDa).-UAS$Kek5∆2GFP-did-not-appear-on-the-western-blot.-However,-

given- that- these- cell- lysates- were- taken- at- day- 6- post$transfection,- a- time- point- at- cells-

expressing- UAS$Kek5∆2GFP-were- undergoing- death- and- had- already- ceased- giving- off- a-

GFP-signal,-the-absence-of-a-signal-for-the-∆2-variant-was-expected.-

,, ,

UAS-Kek5

Δ3 GFP

UAS-Kek5

Δ2 GFP

UAS-Kek5

wt GFP

124 kDa

130-kDa

25-

!

DISCUSSION,#

If- cells- cannot- communicate- with- one- another,- than- a- cohesive- multicellular-

organism- cannot- develop.- Cell- signaling- is- a- crucial- aspect- of- the- development- of- all-

animals,-and-while-there-are-many-pathways-throughout-all-existing-species,-many-of-these-

pathways- are- highly- conserved.- One- such- highly$conserved- pathway- is- the- Bone-

Morphogenetic-Protein-(BMP)-pathway,-which-influences-the-patterning-and-development-

of-many-different- types- of- tissues- from-Drosophila# to- humans.-Dysregulation- of- the-BMP-

pathway- can- result- in- the- generation- of- tumors- and- other- diseases- in- humans- (Little- &-

Mullins,-2006).-Because-the-BMP-signaling-pathway-is-widely-conserved,-the-more-insight-

that-can-be-gathered-on-this-pathway-and-how-it-is-regulated,-the-better.--

In- the- past,- the- intracellular- region- of- Kek5- has- been- demonstrated- to- play- some-

role-in-regulating-BMP-signaling-and-to-influence-certain-lethal-cell-phenotypes.-The-second-

and- third-SLiMs-of- the- intercellular- region-of-Kek5- in-particular,- IC2-and- IC3,- seem-to-be-

important-in-regulating-some-aspects-of-BMP-signaling,-as-their-deletion-in-misexpression-

studies-seems-to-influence-a-higher-rate-of-scutellar-bristle-duplication-in-adult-flies-and-IC2-

and-IC3-have-also-been-shown-to-be-key-in-the-development-of-the-lethal-cell-extrusion-and-

large- cell-phenotypes-observed-upon-Kek5-misexpression- in- the-3rd- instar-wing- imaginal-

disc-(Menon,-2013).-Despite-their-apparent-significance-and-the-large-array-of-Kek5-IC-SLiM-

deletion-variants-that-have-been-generated-in-the-past,-their-respective-roles-are-unknown,-

as-individual-deletion-variants-have-not-yet-been-generated.-Therefore,-in-order-to-allow-for-

the-determination-the-significance-and-specific-functions-of-IC2-and-IC3,-a-small-analysis-of-

IC2- and- IC3- conservation- was- undertaken- and- individual- deletion- variants,- Kek5∆2- and-

Kek5∆3,-were-generated.-

- The-results-of-the-phylogenetic-analysis-revealed-that,-within-Hexapoda,-sequences-

representative-of-IC2-and-IC3-are-highly-conserved-within-Kek5-homologs.-Even-in-Daphnia#

pulex,-a-member-of-Crustacea-separated-evolutionarily-from-Hexapoda-by-millions-of-years,-

IC2-and-IC3-are-still-present-within-this-species’-homolog-of-Kek5.-This-large-time-span-of-

conservation-suggests-that,-whatever-their-function-may-be,-IC2-and-IC3-are-indispensable-

for-Kek5-function.-However,-while-Kek5-orthologs-were-searched-for-in-other-members-of-

26-

!

Crustacea- and- members- of- Chelicerata,- there- is- not- much- data- on- orthologous- protein-

sequences-in-general-for-these-species.-In-the-future,-this-study-may-produce-results-more-

representative- of- Kek5- IC2- and- IC3- conservation- in- Arthropoda- species- outside- of-

Hexapoda.--

- Based-on- the- sequencing- information- for- each- construct,- and- the- expression-data,-

the- generation- of- expressible- Kek5∆2# and# Kek5∆3- constructs- was- successful.- While- co$

transfected- cells- expressing-Kek5∆2-exhibited- increased-death-by-day-5-post$transfection,-

the-microscopy-images-taken-on-day-2-post$transfection-suggest-that-Kek5∆2$GFP-is-being-

expressed- and- is- localizing- to- the- cell-membrane-much- like-Kek5wt$GFP-and-Kek5∆3$GFP.-

However,-a-western-blot-of-Kek5∆2$GFP-at-earlier-times-points-have-yet-to-be-performed.-In-

a- future-experiment,- a-new-co$transfection-of-ArmIGal4#and-pUASTaKek5∆2GFP- should-be-

set- up,- and- the- cells- from- that- co$transfection- should- be- harvested- around- day- 2- post$

transfection-for-use-in-a-Western-blot-to-confirm-expression-and-the-size-of-this-protein.-At-

the- same- time,- a-more- closely-monitored- time- course- should- be- taken- of- the-Kek5∆2- co$

transfection-to-gather-more-information-on-the-transition-from-cells-expressing-Kek5∆2$GFP-

to-cells-dying-off.-In-this-way,-it-can-be-determined-whether-or-not-the-observed-cell-death-is-

linked-to-the-expression-of-Kek5∆2$GFP-and-how-this-cell-death-is-occurring.--

- However,-while-Kek5∆2$GFP- could-not- be- visualized-on- a-Western-blot- due- to- cell-

death- and- subsequent- protein- degredation,- the- cell- death- seen- upon- expression- of- this-

protein-is-a-highly-interesting-result.-The-stark-contrast-between-the-high-rates-of-cell-death-

seen- in- the-UASIKek5∆2IGFP- co$transfection-and- the-healthy- cells- seen- in- the-UASIKek5∆3I

GFP# co$transfection- provides- the- first- set- of- evidence- for- IC2- and- IC3- having- distinct-

functions- from- one- another.- Based- on- the- increased- cell- death- seen- in- cells- expressing-

Kek5∆2$GFP,-IC2-seems-to-be-more-crucial-to-Kek5-function,-and-the-previously-suggested-

notion-that-this-region-is-a-regulator-of-Kek5-itself-is-supported-by-the-data-presented-here.-

- The- generation- of- stable- stocks- of- pUASTaKek5∆2GFP# and# pUASTaKek5∆3GFP# is- a-

crucial-aspect-of-determining-IC2-and-IC3-function-in#vivo.-So-far,-F1-transgenics-have-been-

successfully- gathered- from- the- G0- crosses- set- up- with- w1118- from- the- original- injected-

larvae.-These-F1- transgenics,- signified-by- the-presence-of-eye- color,-have-been-crossed- to-

w1118- and- the- lab- is- still- awaiting- the- F2- generation- from- these- crosses.- Once- the- F2-

generation- is-produced,- the-chromosomal- location-of- the-Kek5∆2- or-Kek5∆3- gene- insertion-

27-

!

can-be-mapped-and-stable-stocks-can-be-generated.-These-stable-stocks-can-be-crossed-to-

A9.Gal4- or-Ptc.Gal4- flies- to- initiate-Kek5∆2- or-Kek5∆3- expression- in- the-offspring-of- such-a-

cross.- While- this- means- that- misexpression- studies- for- Kek5∆2- or- Kek5∆3- in- Drosophila#

melanogaster-are-still-a-few-months-off,-the-crucial-first-steps-in-this-process-have-already-

been-taken.-

In-past-studies,-cell-culture-has-not-been-used-as-a-tool-for-observing-misexpression-

effects- of- Kekkon5- and- its- many- deletion- variants.- This- study- has- shown- that- co$

transfection- of- UASIKek5IGFP# constructs- with- a- Gal4- driver- into- Drosophila# cells- can-

provide- a- relatively-quick- and- easy-method- for- assessing- some- functionalities- associated-

with-SLiMs-of-Kek5,-especially-compared-to-the-long-process-of-generating-stable-transgenic-

lines-in-Drosophila#melanogaster.-

- With-Kek5-playing-a-regulatory-role-in-BMP-signaling,-a-pathway-conserved-in-

vertebrates-that-influences-the-development-of-healthy-tissues-and-cancerous-cells-alike,-

the-characterization-of-Kek5-has-implications-for-combating-BMP$related-diseases-more-

effectively-and-better-understanding-vertebrate-development.-With-constructs-deleting-IC2-

and-IC3-of-Kek5-successfully-generated,-and-a-cell-death-activity-correlated-with-the-

deletion-of-the-SLiM-$-IC2,-a-key-link-between-the-intracellular-structure-and-sequence-of-

Kek5-to-it’s-function-has-been-elucidated.--With-the-extensive-array-of-intracellular-variants-

and-tools-to-assess-cell-death-pathways-readily-available,-rapid-progress-on-defining-the-

mechanism-underlying-the-role-of-IC2,-and-ultimately-Kek5,-in-cell-death/viability-can-now-

be-made.-

-

28-

!

REFERENCES,-

Berg-J.M.,-Tymoczko,-J.L.,-and-Stryer-L.-(2002).-Biochemistry.-New-York:-W.-H.-Freeman-and-Company.-

Bork,-P.,-Holm-L.-and-Sander,-C.-(1994).-The-Immunoglobulin-Fold:-Structural-Classification,-Sequence-Patterns-and-Common-Core.-J.#Mol.#Biol.#242,-309$320.-

Chen,-Y.,-Aulia,-S.,-Li,-L.-and-Tang,-B.L.-(2006).-AMIGO-And-Friends:-An-Emerging-Family-of-Brain$Enriched,-Neuronal-Growth-Modulating,-Type-I-Transmembrane-Proteins-with-Leucine$Rich-repeats-(LRR)-and-Cell-Adhesion-Molecule-Motifs.-Brain#Res.#Rev.#51,-265$274.-

Davey,-N.E.,-Sheilds,-D.C.,-and-Edwards,-R.J.-(2006).-SLiMDisc:-Short,-Linear-Motif-Discovery,-Correcting-for-Common-Evolutionary-Descent.-Nucleic#Acids#Res.#34,-3546$3554.--

Duffy,-J.B.-(2002).-GAL4-System-in-Drosophila:-A-Fly-Geneticist’s-Swiss-Army-Knife.-Genesis#34,-1$15.-

Evans,-T.-(2006).-Characterization-of-Kekkon5,-a-Drosophila#LIG-Protein-that-Modulates-BMP-and-Integrin-Function.-PhD-thesis.-Indiana-University.-

Evans,-T.,-Haridas,-H.,-and-Duffy,-J.B.-(2009).-Kekkon5-is-an-Extracellular-Regulator-of-BMP-Signaling.-Dev.#Biol.-326,-36$46.-

Klueg,-K.M.,-Alvarado,-D.,-Muskavitch,-M.A.T.,-Duffy,-J.B.-(2002).-Creation-of-a-GAL4/UAS$Coupled-Inducible-Gene-Expression-System-for-Use-in-Drosophila-Cultured-Cell-Lines.-Genesis#34,-119–122.-

Ko,-J.-and-Kim,-E.-(2007).-Leucine$Rich-Repeat-Proteins-of-Synapses.-J.#Neurosci.#Res.#85,-2824$2832.-

Little,-S.-and-Mullins,-M.-(2006).-Extracellular-Modulation-of-BMP-Activity-in-Patterning-the-Dorsoventral-Axis.-Birth#Defects#Res.,#Part#C#78,-224$242.-

MacLaren,-C.M.,-Evans,-T.A.,-Alvarado,-D.,-and-Duffy,-J.B.-(2004).-Comparative-Analysis-of-the-Kekkon-Molecules,-Related-Members-of-the-LIG-Superfamily.-Dev.#Genes#Evol.#214,-360$366.-

Mandai,-K.,-Guo,-T.,-St-Hillaire,-C.,-Meabon,-J.S.,-Kanning,-K.C.,-Bothwell,-M.,-and-Ginty,-D.D.-(2009).-LIG-Family-Receptor-Tyrosine-Kinase$Associated-Proteins-Modulate-Growth-Factor-Signals-during-Neural-Development.-Neuron#63,-614$627.-

Menon,-H.-(2013).-Deciphering-the-Role-of-Kekkon5-in-BMP-Signaling-and-Cell-Junction-Biology.-PhD-thesis.-Worcester-Polytechnic-Institute.--

29-

!

Musacchio,-M.,-and-Perrimon,-N.-(1996).-The-Drosophila-Kekkon-Genes:-Novel-Members-of-Both-the-Leucine$Rich-Repeat-and-Immunoglobulin-Superfamilies-Expressed-in-the-CNS.-Dev.#Biol.#178,-63$76.-

Ng,-A.C.,-Eisenberg,-J.M.,-Heath,-R.J.,-Huett,-A.,-Robinson,-C.M.,-Nau,-G.J.,-and-Xavier,-R.J.-(2010).-Human-Leucine$Rich-Repeat-Proteins:-A-Genome$Wide-Bioinformatic-Categorization-and-Functional-Analysis-in-Innate-Immunity.-Proc.#Natl.#Acad.#Sci.#USA#108,-Suppl.#1,-4631$4638.-

Padmanabhan,-M.,-Cournoyer,-P.,-and-Dinesh$Kumar,-S.P.-(2009).-The-Leucine$Rich-Repeat-Domain-in-Plant-Innate-Immunity:-A-Wealth-of-Possibilities.-Cell#Microbiol.#11,-191$198.-

-

30-

!

Appendix,A:,Site\Directed,Mutagenesis,Primer,Sequences,and,Locations,,,Primer#List#,

Primer,Name, Sequence, Description,W500- TAC-GGC-ATC-CGT-TCG-CCA-CCG-TCG-C- 5’-IC2-Flank-W501- TGG-CAA-GCT-ATT-GGC-ATA-CGC-ATC- 3’-IC2-Flank-W502- GAG-CCA-GTG-TAC-GAC-AAC-TTG-GGA-TTG-C- 5’-IC3-Flank-W503- CAC-ACC-CGT-GGC-CGG-CGA-ATA-GAC- 3’-IC3-Flank-

,Primer#Position#on#Kek5wt#

,,, ,

31-

!

Appendix,B:,Ongoing,Transgenic,F1,Crosses,in,Drosophila,,,

Kek5∆2,Transgenic,F1,Crosses, Kek5∆3,Transgenic,F1,Crosses,1A$2F$1M- 2A$11F$1F-1A$2F$2F- 2A$11F$2M-1A$4F$1M- 2A$16F$1F-1A$4F$2M- 2A$16F$2M-1A$4F$3M- 2A$16F$3M-1A$8M$1F- 2A$27F$1M-1A$8M$2M- 2A$27F$2F-1A$8M$3M- 2A$34F$1M-1A$9F$1F- 2A$34F$2F-1A$18M$1M- 2A$4F$1M-1A$18M$2M- 2A$18F$1F-1B$2F$1M- 2A$18F$2M-1B$2F$2M- 2A$22M$1M-1B$12M$1M- 2A$22M$2M-1B$12M$2F- 2B$4F$1M-- 2B$4F$2M-- 2B$6M$1F-- 2B$6M$2F-- 2C$25M$1M-, 2C$25M$2M-, 2C$29F$1M-,Notes:%All-lines-were-crossed-with-w1118-flies-of-the-opposite-sex.-The-first-section-of-each-transgenic-label-corresponds-to-the-vial-each-G0-originated-from.-The-second-section-of-each-label-corresponds-to-the-sex-of-the-parent-G0-and-the-order-in-which-it-eclosed-compared-to-its-siblings.-The-third-section-of-each-label-corresponds-to-the-sex-of-each-F1-transgenic-and-the-order-in-which-it-eclosed.--,,,,, ,

32-

!

Appendix,C:,GENE,CONSTRUCTION,CONSTRUCTS,,

CGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG

CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT

TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCAAAAAGGCCA

TCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTT

GCTTCACAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACG

AAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCGTTAACGCTAGCATG

GATCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAAT

TGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCTCCAGGAAA ATG

ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG GCC CTA CCG CCG CCC ACC GCA GGC

ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC CAC TGT CAG TGG AAT TCG GGC AAG AAG

CTCGGAAAGCAAAATAAACTACGGACCGTCAAGGGATGAGAGCGCAATTGCGATCGTAC

CTAGAGCCCGGGGTTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTTTA

ACTACTCGTTACGAAAAAATATTACGGTTGAAACATGTTTTTTCGTCCGAGGTCCTTT TAC

TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC CGG GAT GGC GGC GGG TGG CGT CCG

TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG GTG ACA GTC ACC TTA AGC CCG TTC TTC

GCGGACCATAGAAATATCAGGACAGCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCC

GCCTCGGATACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGAGTGTACAAGA

AAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGATCGGTCCTTCTCAAACATCTTTGCGTTTTTCCGGT

AGGCAGTCCTACCGGAAGACGAATCAAACTACGGACCGTCAAATACCGCCCGCAGGACGGGCGGTGGGAGGCCCGGCAA

CGAAGTGTTGCAAGTTTAGGCGAGGGCCGCCTAAACAGGATGAGTCCTCTCGCAAGTGGCTGTTTGTTGTCTATTTTGC

TTTCCGGGTCAGAAGGCTGA

M

I L L L L G V L V V L M A L P P P T A G

T T D W M Q S C G T C H C Q W N S G K K

S A D C K N K A L T K I P Q D M S N E M

Q V L D F A H N Q I P E L R R E E F L L

A G L P N V H K I F L R N C T I Q E V H

R E A F K G L H I L I E L D L S G N R I

R E L H P G T F A G L E K L R N V I I N

N N E I E V L P N H L F V N L S F L S R

I E F R N N R L R Q V Q L H V F A G T M

A L S A I S L E Q N R L S H L H K E T F

K D L Q K L M H L S L Q G N A W N C S C

E L Q D F R D F A I S K R L Y T P P T D

C Q E P P Q L R G K L W S E V P S E N F

A C R P R I L G S V R S F I E A N H D N

I S L P C R I V G S P R P N V T W V Y N

K R P L Q Q Y D P R V R V L T S V E Q M

P E Q P S Q V L T S E L R I V G V R A S

D K G A Y T C V A D N R G G R A E A E F

Q L L V S G D Y A G A V S A S D G M G M

G A I G A P T I D P Q T N M F L I I C L

I I T T L L L L L L V A V L T L F W Y C

R R I K T Y Q K D T T M M S G D G L I S

S K M D K T H N G S M L E G S V I M E M

Q K S L L N E V N P V E K P P R R T D I

E S V D G G D D V L E I K K T L L D D T

V Y V A N H S R D E E A V S V A M S D T

T T T P R S R H T Y V D D A Y A N S L P

Y G I R S P P S L T S P V Y T H M T P H

G I Y G T K T M T A P H N G F M T L Q H

P K S R N L A L I A T T N S S R Q H Q H

H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q

Q Q Q Q Q H P L A T T S P F L P A P V V

Y S P A T G V V M K Q G Y M T I P R K P

R A P S W A P S T S G A A G H G S I Q L

S E F Q S P T S P N P S E T G T A T T A

E L Q A E P V Y D N L G L R T T A G G N

S T L N L T K I A G S Q G G A G Q Q Y S

M R D R P L P A T P S L T S V S S A T N

A S K I Y E P I H E L I Q Q Q Q Q L Q Q

Q Q Q Q Q Q Q R L G S M D T E P L Y G V

R Q Q G I T I L P G S S I S G A G L G H

A A Y L S P G S G A A V S P S H A S S S

G D S P K A A K I P P R P P P K P K K K

M S V T T T R S G Q G S T S Q L F D D E

G E D G T E V

W50 Start of Good Sequence (B1, pENTRK5∆2)

NheI

NheI

attL1

junction markersignal sequence

KEK5

N FLANK

1

80

159

238

317

396

475

537

598

658

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG

CAG GTG CTG GAC TTT GCC CAC AAT CAA ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG

GCC GGT CTG CCC AAT GTG CAC AAG ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT

CGC GAG GCC TTC AAG GGT CTG CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA

CGG GAA CTG CAT CCG GGC ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gTG ATC ATC AAC

AAC AAC GAG ATC GAG GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC

ATC GAG TTC CGG AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG

GCG CTG AGC GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC

TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC

GTC CAC GAC CTG AAA CGG GTG TTA GTT TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC

CGG CCA GAC GGG TTA CAC GTG TTC TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA

GCG CTC CGG AAG TTC CCA GAC GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT

GCC CTT GAC GTA GGC CCG TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cAC TAG TAG TTG

TTG TTG CTC TAG CTC CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG

TAG CTC AAG GCC TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC

CGC GAC TCG CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG

M













































G E D G T E V

LRRs

BglII

Actual sequence is GTG - should match original wt sequence

oligo238

718

778

838

898

958

1018

1078

1138

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

AAG GAT CTG CAG AAG CTG ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC

GAG CTG CAG GAC TTT CGC GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT

TGC CAG GAG CCG CCA CAG CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC TTC

GCC TGC CGG CCG CGC ATT TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC GAC AAT

ATC TCG CTA CCC TGC CGC ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG GTG TAC AAC

AAG CGG CCA TTG CAG CAG TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC GTG GAA CAG ATG

CCG GAG CAG CCC TCC CAG GTG CTC ACC TCG GAG CTG CGC ATC GTG GGC GTA CGG GCC TCC

GAC AAG GGT GCC TAC ACC TGT GTG GCG GAT AAC CGG GGC GGA CGG GCG GAG GCC GAG TTC

TTC CTA GAC GTC TTC GAC TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG

CTC GAC GTC CTG AAA GCG CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA

ACG GTC CTC GGC GGT GTC GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG AAG

CGG ACG GCC GGC GCG TAA AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG CTG TTA

TAG AGC GAT GGG ACG GCG TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC CAC ATG TTG

TTC GCC GGT AAC GTC GTC ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG CAC CTT GTC TAC

GGC CTC GTC GGG AGG GTC CAC GAG TGG AGC CTC GAC GCG TAG CAC CCG CAT GCC CGG AGG

CTG TTC CCA CGG ATG TGG ACA CAC CGC CTA TTG GCC CCG CCT GCC CGC CTC CGG CTC AAG

M













































G E D G T E V

oligo594

C FLANK

Ig

1198

1258

1318

1378

1438

1498

1558

1618

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

CAG CTG CTC GTG AGC GGT GAC TAT GCC GGC GCG GTA TCC GCC TCC GAT GGC ATG GGC ATG

GGC GCC ATT GGG GCA CCA ACC ATT GAT CCG CAA ACG AAC ATG TTT CTC ATC ATC TGT CTA

ATC ATT ACG ACG CTG CTG CTC CTG CTG CTC GTG GCG GTG CTG ACG CTC TTC TGG TAC TGC

CGT CGC ATC AAG ACC TAT CAA AAG GAC ACC ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT

TCC AAG ATG GAC AAG ACG CAC AAC GGC TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG

CAG AAG AGC CTG CTC AAC GAG GTC AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC

GTC GAC GAG CAC TCG CCA CTG ATA CGG CCG CGC CAT AGG CGG AGG CTA CCG TAC CCG TAC

CCG CGG TAA CCC CGT GGT TGG TAA CTA GGC GTT TGC TTG TAC AAA GAG TAG TAG ACA GAT

TAG TAA TGC TGC GAC GAC GAG GAC GAC GAG CAC CGC CAC GAC TGC GAG AAG ACC ATG ACG

GCA GCG TAG TTC TGG ATA GTT TTC CTG TGG TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA

AGG TTC TAC CTG TTC TGC GTG TTG CCG AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC

GTC TTC TCG GAC GAG TTG CTC CAG TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG

M













































G E D G T E V

end Ig?

Tm

IC

∆123 Confirmed∆1235 Confirmed∆1234 Confirmed

oligo745 apK5_seq_int_5'

IC1 modified


∆1 ConfirmedIC1

1678

1738

1798

1858

1918

1978

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

GAG AGC GTG GAT GGT GGC GAT GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC

GTC TAT GTG GCC AAT CAC TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG

ACG ACC ACG CCC CGA TCT CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA

TAC GGC ATC CGT TCG CCA CCG TCG CTA ACC AGT CCG GTC TAC ACG CAT ATG ACG CCG CAC

GGC ATC TAC GGC ACC AAG ACG ATG ACG GCT CCG CAT AAC GGC TTT ATG ACG CTG CAG CAT

CCC AAG TCG CGC AAC CTG GCG CTC ATT GCC ACC ACC AAC AGC AGT CGC CAG CAC CAG CAC

CAC CAT CAG CTG CAG CAG CAG CAG CAG CAC CAC CAC CAC CAC CAG CAG CAA CAA CAA CAG

CAG CAG CAG CAG CAA CAT CCG CTG GCC ACC ACA TCG CCC TTC CTG CCC GCA CCC GTC GTC

CTC TCG CAC CTA CCA CCG CTA CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG

CAG ATA CAC CGG TTA GTG AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC

TGC TGG TGC GGG GCT AGA GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT

ATG CCG TAG GCA AGC GGT GGC AGC GAT TGG TCA GGC CAG ATG TGC GTA TAC TGC GGC GTG

CCG TAG ATG CCG TGG TTC TGC TAC TGC CGA GGC GTA TTG CCG AAA TAC TGC GAC GTC GTA

GGG TTC AGC GCG TTG GAC CGC GAG TAA CGG TGG TGG TTG TCG TCA GCG GTC GTG GTC GTG

GTG GTA GTC GAC GTC GTC GTC GTC GTC GTG GTG GTG GTG GTG GTC GTC GTT GTT GTT GTC

GTC GTC GTC GTC GTT GTA GGC GAC CGG TGG TGT AGC GGG AAG GAC GGG CGT GGG CAG CAG

M













































G E D G T E V

oligo240

∆IC2 ChloeL

2038

2098

2158

2218

2278

2338

2398

2458

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

TAT TCG CCG GCC ACG GGT GTG GTC ATG AAA CAG GGA TAT ATG ACC ATT CCG CGC AAG CCG

CGC GCT CCC AGC TGG GCG CCC AGT ACT TCC GGT GCC GCT GGC CAC GGA TCC ATT CAG CTA

AGT GAA TTC CAG AGC CCC ACA TCG CCG AAT CCC AGC GAG ACT GGC ACC GCC ACC ACC GCG

GAA CTG CAG GCG GAG CCA GTG TAC GAC AAC TTG GGA TTG CGA ACC ACT GCC GGC GGC AAC

TCC ACC CTC AAT CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC GCT GGT CAG CAG TAC TCG

ATG CGG GAC CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA TCG GTG TCC TCG GCG ACC AAT

GCC AGT AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT CAG CAG CAA CAG CAG TTG CAA CAA

ATA AGC GGC CGG TGC CCA CAC CAG TAC TTT GTC CCT ATA TAC TGG TAA GGC GCG TTC GGC

GCG CGA GGG TCG ACC CGC GGG TCA TGA AGG CCA CGG CGA CCG GTG CCT AGG TAA GTC GAT

TCA CTT AAG GTC TCG GGG TGT AGC GGC TTA GGG TCG CTC TGA CCG TGG CGG TGG TGG CGC

CTT GAC GTC CGC CTC GGT CAC ATG CTG TTG AAC CCT AAC GCT TGG TGA CGG CCG CCG TTG

AGG TGG GAG TTA GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG CGA CCA GTC GTC ATG AGC

TAC GCC CTG GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT AGC CAC AGG AGC CGC TGG TTA

CGG TCA TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA GTC GTC GTT GTC GTC AAC GTT GTT

M













































G E D G T E V

IC3 modified IC3

oligo241

∆45 Confirmed∆4 ConfirmedIC4 modified

IC4

2518

2578

2638

2698

2758

2818

2878

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

CAA CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC ATG GAC ACG GAA CCC CTG TAC GGA GTT

CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC TCG AGC ATT AGC GGT GCC GGA CTG GGC CAC

GCC GCC TAC CTT TCA CCC GGC TCG GGT GCC GCC GTA TCG CCA AGC CAC GCC AGC AGC AGC

GGT GAC TCT CCG AAG GCC GCC AAG ATC CCA CCA CGC CCA CCA CCG AAG CCC AAG AAG AAG

ATG TCC GTG ACG ACG ACG CGC AGC GGC CAG GGC AGC ACC AGC CAG CTC TTC GAC GAC GAG

GGC GAG GAT GGC ACC GAG GTC G ACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAA

AGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATT

ATTTGCCATCCAGCTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT

GTT GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG TAC CTG TGC CTT GGG GAC ATG CCT CAA

GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG AGC TCG TAA TCG CCA CGG CCT GAC CCG GTG

CGG CGG ATG GAA AGT GGG CCG AGC CCA CGG CGG CAT AGC GGT TCG GTG CGG TCG TCG TCG

CCA CTG AGA GGC TTC CGG CGG TTC TAG GGT GGT GCG GGT GGT GGC TTC GGG TTC TTC TTC

TAC AGG CAC TGC TGC TGC GCG TCG CCG GTC CCG TCG TGG TCG GTC GAG AAG CTG CTG CTC

CCG CTC CTA CCG TGG CTC CAG C TGGGTCGAAAGAACATGTTTCAACCGTAATATTCTT

TCGTAACGAATAGTTAAACAACGTTGCTTGTCCAGTGATAGTCAGTTTTATTTTAGTAA

TAAACGGTAGGTCGACGTCGAGACCGGGCACAGAGTTTTAGAGACTACAATGTAACGTGTTCTATTTTTATATAGTA

M













































G E D G T E V

∆1235 Confirmed∆5 ConfirmedIC5

IC6 (PDZ)

attB2attL2

End of Good Sequence (B1, pENTRK5∆2) OLIGO 691

2938

2998

3058

3118

3178

3238

3296

3355

1

2

22

42

62

82

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

882

CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAG

GCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCG

ACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATG

TTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCC

TGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGT

GAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCG

ATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCG

TAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCAT

GGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCG

CAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCA

AAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTG

GTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACG

TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTA

ATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC

CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA

GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT

CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC

CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG

GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAA

GTACTTGTTATTTTGACAGACGAATGTATTTGTCATTATGTTCCCCACAATACTCGGTATAAGTTGCCCTTTGCAGCTC

CGGCGCTAATTTAAGGTTGTACCTACGACTAAATATACCCATATTTACCCGAGCGCTATTACAGCCCGTTAGTCCACGC

TGTTAGATAGCGAACATACCCTTCGGGCTACGCGGTCTCAACAAAGACTTTGTACCGTTTCCATCGCAACGGTTACTAC

AATGTCTACTCTACCAGTCTGATTTGACCGACTGCCTTAAATACGGAGAAGGCTGGTAGTTCGTAAAATAGGCATGAGG

ACTACTACGTACCAATGAGTGGTGACGCTAGGGGCCTTTTTGTCGTAAGGTCCATAATCTTCTTATAGGACTAAGTCCA

CTTTTATAACAACTACGCGACCGTCACAAGGACGCGGCCAACGTAAGCTAAGGACAAACATTAACAGGAAAATTGTCGC

TAGCGCATAAAGCAGAGCGAGTCCGCGTTAGTGCTTACTTATTGCCAAACCAACTACGCTCACTAAAACTACTGCTCGC

ATTACCGACCGGACAACTTGTTCAGACCTTTCTTTACGTATTTGAAAACGGTAAGAGTGGCCTAAGTCAGCAGTGAGTA

CCACTAAAGAGTGAACTATTGGAATAAAAACTGCTCCCCTTTAATTATCCAACATAACTACAACCTGCTCAGCCTTAGC

GTCTGGCTATGGTCCTAGAACGGTAGGATACCTTGACGGAGCCACTCAAAAGAGGAAGTAATGTCTTTGCCGAAAAAGT

TTTTATACCATAACTATTAGGACTATACTTATTTAACGTCAAAGTAAACTACGAGCTACTCAAAAAGATTAGTCTTAAC

CAATTAACCAACATTGTGACCGTCTCGTAATGCGACTGAACTGCCCTGCCGCGTTCGAGTACTGGTTTTAGGGAATTGC

ACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTAGTTTCCTAGAAGAACTCTAGGAAAAAAAGACGCGCAT

TAGACGACGAACGTTTGTTTTTTTGGTGGCGATGGTCGCCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAG

GCTTCCATTGACCGAAGTCGTCTCGCGTCTATGGTTTATGACAGGAAGATCACATCGGCATCAATCCGGTGGTGAAGTT

CTTGAGACATCGTGGCGGATGTATGGAGCGAGACGATTAGGACAATGGTCACCGACGACGGTCACCGCTATTCAGCACA

GAATGGCCCAACCTGAGTTCTGCTATCAATGGCCTATTCCGCGTCGCCAGCCCGACTTGCCCCCCAAGCACGTGTGTCG

GGTCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGTCGCACTCGATACTCTTTCGCGGTGCGAAGGGCTTCC

CTCTTTCCGCCTGTCCATAGGCCATTCGCCGTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTT

3432

3511

3590

3669

3748

3827

3906

3985

4064

4143

4222

4301

4380

4459

4538

4617

4696

4775

4854

CCA TCC AGC TGC AGC TCT GGC CCG TGT CTC AAA ATC TCT GAT GTT ACA TTG CAC AAG ATA

AAA ATA TAT CAT CAT GAA CAA TAA AAC TGT CTG CTT ACA TAA ACA GTA ATA CAA GGG GTG

TTA TGA GCC ATA TTC AAC GGG AAA CGT CGA GGC CGC GAT TAA ATT CCA ACA TGG ATG CTG

ATT TAT ATG GGT ATA AAT GGG CTC GCG ATA ATG TCG GGC AAT CAG GTG CGA CAA TCT ATC

GCT TGT ATG GGA AGC CCG ATG CGC CAG AGT TGT TTC TGA AAC ATG GCA AAG GTA GCG TTG

CCA ATG ATG TTA CAG ATG AGA TGG TCA GAC TAA ACT GGC TGA CGG AAT TTA TGC CTC TTC

CGA CCA TCA AGC ATT TTA TCC GTA CTC CTG ATG ATG CAT GGT TAC TCA CCA CTG CGA TCC

CCG GAA AAA CAG CAT TCC AGG TAT TAG AAG AAT ATC CTG ATT CAG GTG AAA ATA TTG TTG

ATG CGC TGG CAG TGT TCC TGC GCC GGT TGC ATT CGA TTC CTG TTT GTA ATT GTC CTT TTA

ACA GCG ATC GCG TAT TTC GTC TCG CTC AGG CGC AAT CAC GAA TGA ATA ACG GTT TGG TTG

ATG CGA GTG ATT TTG ATG ACG AGC GTA ATG GCT GGC CTG TTG AAC AAG TCT GGA AAG AAA

TGC ATA AAC TTT TGC CAT TCT CAC CGG ATT CAG TCG TCA CTC ATG GTG ATT TCT CAC TTG

ATA ACC TTA TTT TTG ACG AGG GGA AAT TAA TAG GTT GTA TTG ATG TTG GAC GAG TCG GAA

TCG CAG ACC GAT ACC AGG ATC TTG CCA TCC TAT GGA ACT GCC TCG GTG AGT TTT CTC CTT

CAT TAC AGA AAC GGC TTT TTC AAA AAT ATG GTA TTG ATA ATC CTG ATA TGA ATA AAT TGC

AGT TTC ATT TGA TGC TCG ATG AGT TTT TCT AAT CAG AAT TGG TTA ATT GGT TGT AAC ACT

GGC AGA GCA TTA CGC TGA CTT GAC GGG ACG GCG CAA GCT CAT GAC CAA AAT CCC TTA ACG

TGA GTT TTC GTT CCA CTG AGC GTC AGA CCC CGT AGA AAA GAT CAA AGG ATC TTC TTG AGA

TCC TTT TTT TCT GCG CGT AAT CTG CTG CTT GCA AAC AAA AAA ACC ACC GCT ACC AGC GGT

GGT TTG TTT GCC GGA TCA AGA GCT ACC AAC TCT TTT TCC GAA GGT AAC TGG CTT CAG CAG

AGC GCA GAT ACC AAA TAC TGT CCT TCT AGT GTA GCC GTA GTT AGG CCA CCA CTT CAA GAA

GGT AGG TCG ACG TCG AGA CCG GGC ACA GAG TTT TAG AGA CTA CAA TGT AAC GTG TTC TAT

TTT TAT ATA GTA GTA CTT GTT ATT TTG ACA GAC GAA TGT ATT TGT CAT TAT GTT CCC CAC

AAT ACT CGG TAT AAG TTG CCC TTT GCA GCT CCG GCG CTA ATT TAA GGT TGT ACC TAC GAC

TAA ATA TAC CCA TAT TTA CCC GAG CGC TAT TAC AGC CCG TTA GTC CAC GCT GTT AGA TAG

CGA ACA TAC CCT TCG GGC TAC GCG GTC TCA ACA AAG ACT TTG TAC CGT TTC CAT CGC AAC

GGT TAC TAC AAT GTC TAC TCT ACC AGT CTG ATT TGA CCG ACT GCC TTA AAT ACG GAG AAG

GCT GGT AGT TCG TAA AAT AGG CAT GAG GAC TAC TAC GTA CCA ATG AGT GGT GAC GCT AGG

GGC CTT TTT GTC GTA AGG TCC ATA ATC TTC TTA TAG GAC TAA GTC CAC TTT TAT AAC AAC

TAC GCG ACC GTC ACA AGG ACG CGG CCA ACG TAA GCT AAG GAC AAA CAT TAA CAG GAA AAT

TGT CGC TAG CGC ATA AAG CAG AGC GAG TCC GCG TTA GTG CTT ACT TAT TGC CAA ACC AAC

TAC GCT CAC TAA AAC TAC TGC TCG CAT TAC CGA CCG GAC AAC TTG TTC AGA CCT TTC TTT

ACG TAT TTG AAA ACG GTA AGA GTG GCC TAA GTC AGC AGT GAG TAC CAC TAA AGA GTG AAC

TAT TGG AAT AAA AAC TGC TCC CCT TTA ATT ATC CAA CAT AAC TAC AAC CTG CTC AGC CTT

AGC GTC TGG CTA TGG TCC TAG AAC GGT AGG ATA CCT TGA CGG AGC CAC TCA AAA GAG GAA

GTA ATG TCT TTG CCG AAA AAG TTT TTA TAC CAT AAC TAT TAG GAC TAT ACT TAT TTA ACG

TCA AAG TAA ACT ACG AGC TAC TCA AAA AGA TTA GTC TTA ACC AAT TAA CCA ACA TTG TGA

CCG TCT CGT AAT GCG ACT GAA CTG CCC TGC CGC GTT CGA GTA CTG GTT TTA GGG AAT TGC

ACT CAA AAG CAA GGT GAC TCG CAG TCT GGG GCA TCT TTT CTA GTT TCC TAG AAG AAC TCT

AGG AAA AAA AGA CGC GCA TTA GAC GAC GAA CGT TTG TTT TTT TGG TGG CGA TGG TCG CCA

CCA AAC AAA CGG CCT AGT TCT CGA TGG TTG AGA AAA AGG CTT CCA TTG ACC GAA GTC GTC

TCG CGT CTA TGG TTT ATG ACA GGA AGA TCA CAT CGG CAT CAA TCC GGT GGT GAA GTT CTT

1

61

121

181

241

301

361

421

481

541

601

661

721

781

841

901

961

1021

1081

1141

1201

CTC TGT AGC ACC GCC TAC ATA CCT CGC TCT GCT AAT CCT GTT ACC AGT GGC TGC TGC CAG

TGG CGA TAA GTC GTG TCT TAC CGG GTT GGA CTC AAG ACG ATA GTT ACC GGA TAA GGC GCA

GCG GTC GGG CTG AAC GGG GGG TTC GTG CAC ACA GCC CAG CTT GGA GCG AAC GAC CTA CAC

CGA ACT GAG ATA CCT ACA GCG TGA GCT ATG AGA AAG CGC CAC GCT TCC CGA AGG GAG AAA

GGC GGA CAG GTA TCC GGT AAG CGG CAG GGT CGG AAC AGG AGA GCG CAC GAG GGA GCT TCC

AGG GGG AAA CGC CTG GTA TCT TTA TAG TCC TGT CGG GTT TCG CCA CCT CTG ACT TGA GCG

TCG ATT TTT GTG ATG CTC GTC AGG GGG GCG GAG CCT ATG GAA AAA CGC CAG CAA CGC GGC

CTT TTT ACG GTT CCT GGC CTT TTG CTG GCC TTT TGC TCA CAT GTT CTT TCC TGC GTT ATC

CCC TGA TTC TGT GGA TAA CCG TAT TAC CGC TAG CCA GGA AGA GTT TGT AGA AAC GCA AAA

AGG CCA TCC GTC AGG ATG GCC TTC TGC TTA GTT TGA TGC CTG GCA GTT TAT GGC GGG CGT

CCT GCC CGC CAC CCT CCG GGC CGT TGC TTC ACA ACG TTC AAA TCC GCT CCC GGC GGA TTT

GTC CTA CTC AGG AGA GCG TTC ACC GAC AAA CAA CAG ATA AAA CGA AAG GCC CAG TCT TCC

GAC TGA GCC TTT CGT TTT ATT TGA TGC CTG GCA GTT CCC TAC TCT CGC GTT AAC GCT AGC

ATG GAT CTC GGG CCC CAA ATA ATG ATT TTA TTT TGA CTG ATA GTG ACC

TGT TCG TTG CAA CAA ATT GAT GAG CAA TGC TTT TTT ATA ATG CCA

ACT TTG TAC AAA AAA GCA GGC TCC AGG AAA ATG ATC CTT CTG CTG CTG GGT

CT CGG AAA GCA AAA TAA ACT ACG GAC CGT CAA GGG ATG AGA GCG CAA TTG CGA TCG

TAC CTA GAG CCC GGG GTT TAT TAC TAA AAT AAA ACT GAC TAT CAC TGG

ACA AGC AAC GTT GTT TAA CTA CTC GTT ACG AAA AAA TAT TAC GGT

TGA AAC ATG TTT TTT CGT CCG AGG TCC TTT TAC TAG GAA GAC GAC GAC CCA

GAG ACA TCG TGG CGG ATG TAT GGA GCG AGA CGA TTA GGA CAA TGG TCA CCG ACG ACG GTC

ACC GCT ATT CAG CAC AGA ATG GCC CAA CCT GAG TTC TGC TAT CAA TGG CCT ATT CCG CGT

CGC CAG CCC GAC TTG CCC CCC AAG CAC GTG TGT CGG GTC GAA CCT CGC TTG CTG GAT GTG

GCT TGA CTC TAT GGA TGT CGC ACT CGA TAC TCT TTC GCG GTG CGA AGG GCT TCC CTC TTT

CCG CCT GTC CAT AGG CCA TTC GCC GTC CCA GCC TTG TCC TCT CGC GTG CTC CCT CGA AGG

TCC CCC TTT GCG GAC CAT AGA AAT ATC AGG ACA GCC CAA AGC GGT GGA GAC TGA ACT CGC

AGC TAA AAA CAC TAC GAG CAG TCC CCC CGC CTC GGA TAC CTT TTT GCG GTC GTT GCG CCG

GAA AAA TGC CAA GGA CCG GAA AAC GAC CGG AAA ACG AGT GTA CAA GAA AGG ACG CAA TAG

GGG ACT AAG ACA CCT ATT GGC ATA ATG GCG ATC GGT CCT TCT CAA ACA TCT TTG CGT TTT

TCC GGT AGG CAG TCC TAC CGG AAG ACG AAT CAA ACT ACG GAC CGT CAA ATA CCG CCC GCA

GGA CGG GCG GTG GGA GGC CCG GCA ACG AAG TGT TGC AAG TTT AGG CGA GGG CCG CCT AAA

CAG GAT GAG TCC TCT CGC AAG TGG CTG TTT GTT GTC TAT TTT GCT TTC CGG GTC AGA AGG

CTG A

M I L L L L G

V L V V L M A L P P P T A G T T D W M Q

S C G T C H C Q W N S G K K S A D C K N

K A L T K I P Q D M S N E M Q V L D F A

H N Q I P E L R R E E F L L A G L P N V

H K I F L R N C T I Q E V H R E A F K G

L H I L I E L D L S G N R I R E L H P G

T F A G L E K L R N V I I N N N E I E V

L P N H L F V N L S F L S R I E F R N N

R L R Q V Q L H V F A G T M A L S A I S

L E Q N R L S H L H K E T F K D L Q K L

M H L S L Q G N A W N C S C E L Q D F R

D F A I S K R L Y T P P T D C Q E P P Q

L R G K L W S E V P S E N F A C R P R I

L G S V R S F I E A N H D N I S L P C R

I V G S P R P N V T W V Y N K R P L Q Q

Y D P R V R V L T S V E Q M P E Q P S Q

V L T S E L R I V G V R A S D K G A Y T

C V A D N R G G R A E A E F Q L L V S G

D Y A G A V S A S D G M G M G A I G A P

T I D P Q T N M F L I I C L I I T T L L

L L L L V A V L T L F W Y C R R I K T Y

Q K D T T M M S G D G L I S S K M D K T

H N G S M L E G S V I M E M Q K S L L N

E V N P V E K P P R R T D I E S V D G G

D D V L E I K K T L L D D T V Y V A N H

S R D E E A V S V A M S D T T T T P R S

R H T Y V D D A Y A N S L P P D L L A F

P A R V P P T S P S M Q S S Q S N I P D

Q V I Y G I R S P P S L T S P V Y T H M

T P H G I Y G T K T M T A P H N G F M T

L Q H P K S R N L A L I A T T N S S R Q

H Q H H H Q L Q Q Q Q Q H H H H H Q Q Q

Q Q Q Q Q Q Q Q H P L A T T S P F L P A

P V V Y S P A T G V E P V Y D N L G L R

T T A G G N S T L N L T K I A G S Q G G

A G Q Q Y S M R D R P L P A T P S L T S

V S S A T N A S K I Y E P I H E L I Q Q

Q Q Q L Q Q Q Q Q Q Q Q Q R L G S M D T

E P L Y G V R Q Q G I T I L P G S S I S

G A G L G H A A Y L S P G S G A A V S P

S H A S S S G D S P K A A K I P P R P P

P K P K K K M S V T T T R S G Q G S T S

Q L F D D E G E D G T E V

W50

Start of Good Sequence (D7, pENTRK5∆3)

NheI

NheI

attL1

junction markersignal sequence

KEK5

1261

1321

1381

1441

1501

1561

1621

1681

1741

1801

1861

1921

1981

2041

2089

2134

1

8

28

48

68

88

108

128

148

168

188

208

228

248

268

288

308

328

348

368

388

408

428

448

468

488

508

528

548

568

588

608

628

648

668

688

708

728

748

768

788

808

828

848

GTG CTA GTG GTT CTA ATG GCC CTA CCG CCG CCC ACC GCA GGC ACC ACC GAT TGG ATG CAG

AGC TGC GGT ACA TGC CAC TGT CAG TGG AAT TCG GGC AAG AAG AGC GCC GAC TGC AAG AAC

AAG GCG CTA ACC AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG CAG GTG CTG GAC TTT GCC

CAC AAT CAA ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG GCC GGT CTG CCC AAT GTG

CAC AAG ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT CGC GAG GCC TTC AAG GGT

CTG CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA CGG GAA CTG CAT CCG GGC

ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gTG ATC ATC AAC AAC AAC GAG ATC GAG GTG

CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC ATC GAG TTC CGG AAC AAT

CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG GCG CTG AGC GCC ATT TCG

CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC AAG GAT CTG CAG AAG CTG

ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC GAG CTG CAG GAC TTT CGC

CAC GAT CAC CAA GAT TAC CGG GAT GGC GGC GGG TGG CGT CCG TGG TGG CTA ACC TAC GTC

TCG ACG CCA TGT ACG GTG ACA GTC ACC TTA AGC CCG TTC TTC TCG CGG CTG ACG TTC TTG

TTC CGC GAT TGG TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC GTC CAC GAC CTG AAA CGG

GTG TTA GTT TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC CGG CCA GAC GGG TTA CAC

GTG TTC TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA GCG CTC CGG AAG TTC CCA

GAC GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT GCC CTT GAC GTA GGC CCG

TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cAC TAG TAG TTG TTG TTG CTC TAG CTC CAC

GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG TAG CTC AAG GCC TTG TTA

GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC CGC GAC TCG CGG TAA AGC

GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG TTC CTA GAC GTC TTC GAC

TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG CTC GAC GTC CTG AAA GCG

M I L L L L G












































N FLANK

LRRs

BglII

Actual sequence is GTG - should match original wt sequenceoligo238

oligo594

C FLANK

2185

2245

2305

2365

2425

2485

2545

2605

2665

2725

2785

1

8

28

48

68

88

108

128

148

168

188

208

228

248

268

288

308

328

348

368

388

408

428

448

468

488

508

528

548

568

588

608

628

648

668

688

708

728

748

768

788

808

828

848

GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT TGC CAG GAG CCG CCA CAG

CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC TTC GCC TGC CGG CCG CGC ATT

TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC GAC AAT ATC TCG CTA CCC TGC CGC

ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG GTG TAC AAC AAG CGG CCA TTG CAG CAG

TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC GTG GAA CAG ATG CCG GAG CAG CCC TCC CAG

GTG CTC ACC TCG GAG CTG CGC ATC GTG GGC GTA CGG GCC TCC GAC AAG GGT GCC TAC ACC

TGT GTG GCG GAT AAC CGG GGC GGA CGG GCG GAG GCC GAG TTC CAG CTG CTC GTG AGC GGT

GAC TAT GCC GGC GCG GTA TCC GCC TCC GAT GGC ATG GGC ATG GGC GCC ATT GGG GCA CCA

ACC ATT GAT CCG CAA ACG AAC ATG TTT CTC ATC ATC TGT CTA ATC ATT ACG ACG CTG CTG

CTC CTG CTG CTC GTG GCG GTG CTG ACG CTC TTC TGG TAC TGC CGT CGC ATC AAG ACC TAT

CAA AAG GAC ACC ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT TCC AAG ATG GAC AAG ACG

CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA ACG GTC CTC GGC GGT GTC

GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG AAG CGG ACG GCC GGC GCG TAA

AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG CTG TTA TAG AGC GAT GGG ACG GCG

TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC CAC ATG TTG TTC GCC GGT AAC GTC GTC

ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG CAC CTT GTC TAC GGC CTC GTC GGG AGG GTC

CAC GAG TGG AGC CTC GAC GCG TAG CAC CCG CAT GCC CGG AGG CTG TTC CCA CGG ATG TGG

ACA CAC CGC CTA TTG GCC CCG CCT GCC CGC CTC CGG CTC AAG GTC GAC GAG CAC TCG CCA

CTG ATA CGG CCG CGC CAT AGG CGG AGG CTA CCG TAC CCG TAC CCG CGG TAA CCC CGT GGT

TGG TAA CTA GGC GTT TGC TTG TAC AAA GAG TAG TAG ACA GAT TAG TAA TGC TGC GAC GAC

GAG GAC GAC GAG CAC CGC CAC GAC TGC GAG AAG ACC ATG ACG GCA GCG TAG TTC TGG ATA

GTT TTC CTG TGG TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA AGG TTC TAC CTG TTC TGC

M I L L L L G












































Ig

end Ig?

Tm

IC


2845

2905

2965

3025

3085

3145

3205

3265

3325

3385

3445

1

8

28

48

68

88

108

128

148

168

188

208

228

248

268

288

308

328

348

368

388

408

428

448

468

488

508

528

548

568

588

608

628

648

668

688

708

728

748

768

788

808

828

848

CAC AAC GGC TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG CAG AAG AGC CTG CTC AAC

GAG GTC AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC GAG AGC GTG GAT GGT GGC

GAT GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC GTC TAT GTG GCC AAT CAC

TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG ACG ACC ACG CCC CGA TCT

CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA CCG GAT CTG CTG GCC TTT

CCC GCT CGC GTG CCG CCC ACC TCG CCC TCG ATG CAA TCG TCG CAG TCG AAC ATA CCC GAC

CAG GTG ATC TAC GGC ATC CGT TCG CCA CCG TCG CTA ACC AGT CCG GTC TAC ACG CAT ATG

ACG CCG CAC GGC ATC TAC GGC ACC AAG ACG ATG ACG GCT CCG CAT AAC GGC TTT ATG ACG

CTG CAG CAT CCC AAG TCG CGC AAC CTG GCG CTC ATT GCC ACC ACC AAC AGC AGT CGC CAG

CAC CAG CAC CAC CAT CAG CTG CAG CAG CAG CAG CAG CAC CAC CAC CAC CAC CAG CAG CAA

GTG TTG CCG AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC GTC TTC TCG GAC GAG TTG

CTC CAG TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG CTC TCG CAC CTA CCA CCG

CTA CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG CAG ATA CAC CGG TTA GTG

AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC TGC TGG TGC GGG GCT AGA

GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT GGC CTA GAC GAC CGG AAA

GGG CGA GCG CAC GGC GGG TGG AGC GGG AGC TAC GTT AGC AGC GTC AGC TTG TAT GGG CTG

GTC CAC TAG ATG CCG TAG GCA AGC GGT GGC AGC GAT TGG TCA GGC CAG ATG TGC GTA TAC

TGC GGC GTG CCG TAG ATG CCG TGG TTC TGC TAC TGC CGA GGC GTA TTG CCG AAA TAC TGC

GAC GTC GTA GGG TTC AGC GCG TTG GAC CGC GAG TAA CGG TGG TGG TTG TCG TCA GCG GTC

GTG GTC GTG GTG GTA GTC GAC GTC GTC GTC GTC GTC GTG GTG GTG GTG GTG GTC GTC GTT

M I L L L L G












































IC1 modified oligo746 apK5_seq_int_3'

∆1 ConfirmedIC1

oligo240

∆234 ConfirmedIC2 modified

IC2

3505

3565

3625

3685

3745

3805

3865

3925

3985

4045

1

8

28

48

68

88

108

128

148

168

188

208

228

248

268

288

308

328

348

368

388

408

428

448

468

488

508

528

548

568

588

608

628

648

668

688

708

728

748

768

788

808

828

848

CAA CAA CAG CAG CAG CAG CAG CAA CAT CCG CTG GCC ACC ACA TCG CCC TTC CTG CCC GCA

CCC GTC GTC TAT TCG CCG GCC ACG GGT GTG GAG CCA GTG TAC GAC AAC TTG GGA TTG CGA

ACC ACT GCC GGC GGC AAC TCC ACC CTC AAT CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC

GCT GGT CAG CAG TAC TCG ATG CGG GAC CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA TCG

GTG TCC TCG GCG ACC AAT GCC AGT AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT CAG CAG

CAA CAG CAG TTG CAA CAA CAA CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC ATG GAC ACG

GAA CCC CTG TAC GGA GTT CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC TCG AGC ATT AGC

GGT GCC GGA CTG GGC CAC GCC GCC TAC CTT TCA CCC GGC TCG GGT GCC GCC GTA TCG CCA

AGC CAC GCC AGC AGC AGC GGT GAC TCT CCG AAG GCC GCC AAG ATC CCA CCA CGC CCA CCA

CCG AAG CCC AAG AAG AAG ATG TCC GTG ACG ACG ACG CGC AGC GGC CAG GGC AGC ACC AGC

CAG CTC TTC GAC GAC GAG GGC GAG GAT GGC ACC GAG GTC GAC CCA GCT TTC TTG

GTT GTT GTC GTC GTC GTC GTC GTT GTA GGC GAC CGG TGG TGT AGC GGG AAG GAC GGG CGT

GGG CAG CAG ATA AGC GGC CGG TGC CCA CAC CTC GGT CAC ATG CTG TTG AAC CCT AAC GCT

TGG TGA CGG CCG CCG TTG AGG TGG GAG TTA GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG

CGA CCA GTC GTC ATG AGC TAC GCC CTG GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT AGC

CAC AGG AGC CGC TGG TTA CGG TCA TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA GTC GTC

GTT GTC GTC AAC GTT GTT GTT GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG TAC CTG TGC

CTT GGG GAC ATG CCT CAA GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG AGC TCG TAA TCG

CCA CGG CCT GAC CCG GTG CGG CGG ATG GAA AGT GGG CCG AGC CCA CGG CGG CAT AGC GGT

TCG GTG CGG TCG TCG TCG CCA CTG AGA GGC TTC CGG CGG TTC TAG GGT GGT GCG GGT GGT

GGC TTC GGG TTC TTC TTC TAC AGG CAC TGC TGC TGC GCG TCG CCG GTC CCG TCG TGG TCG

GTC GAG AAG CTG CTG CTC CCG CTC CTA CCG TGG CTC CAG CTG GGT CGA AAG AAC

M I L L L L G












































∆ IC3CLIC4

IC5

IC6 (PDZ) attB2attL2

4105

4165

4225

4285

4345

4405

4465

4525

4585

4645

4705

1

8

28

48

68

88

108

128

148

168

188

208

228

248

268

288

308

328

348

368

388

408

428

448

468

488

508

528

548

568

588

608

628

648

668

688

708

728

748

768

788

808

828

848

TAC AAA GTT GGC ATT ATA AGA AAG CAT TGC TTA TCA ATT TGT TGC

AAC GAA CAG GTC ACT ATC AGT CAA AAT AAA ATC ATT ATT TG

ATG TTT CAA CCG TAA TAT TCT TTC GTA ACG AAT AGT TAA ACA ACG

TTG CTT GTC CAG TGA TAG TCA GTT TTA TTT TAG TAA TAA AC

End of Good Sequence (D7, pENTRK5∆3)

4759

4804

GGCCAGACCCACGTAGTCCAGCGGCAGATCGGCGGCGGAGAAGTTAAGCGTCTCCAGGATGACCTTGCCCGAACTGGG

GCACGTGGTGTTCGACGATGTGCAGCTAATTTCGCCCGGCTCCACGTCCGCCCATTGGTTAATCAGCAGACCCTCGTT

GGCGTAACGGAACCATGAGAGGTACGACAACCATTTGAGGTATACTGGCACCGAGCCCGAGTTCAAGAAGAAGGCGTT

TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT

ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT

GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT

TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA

GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG

CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT

GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT

TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC

TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA

TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGA

GGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG

GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT

AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG

CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC

ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTG

CAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT

GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC

ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAG

AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC

AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA

CCGGTCTGGGTGCATCAGGTCGCCGTCTAGCCGCCGCCTCTTCAATTCGCAGAGGTCCTACTGGAACGGGCTTGACCC

CGTGCACCACAAGCTGCTACACGTCGATTAAAGCGGGCCGAGGTGCAGGCGGGTAACCAATTAGTCGTCTGGGAGCAA

CCGCATTGCCTTGGTACTCTCCATGCTGTTGGTAAACTCCATATGACCGTGGCTCGGGCTCAAGTTCTTCTTCCGCAA

AAAGGTATCCGAGGCGGGGGGACTGCTCGTAGTGTTTTTAGCTGCGAGTTCAGTCTCCACCGCTTTGGGCTGTCCTGA

TATTTCTATGGTCCGCAAAGGGGGACCTTCGAGGGAGCACGCGAGAGGACAAGGCTGGGACGGCGAATGGCCTATGGA

CAGGCGGAAAGAGGGAAGCCCTTCGCACCGCGAAAGAGTTACGAGTGCGACATCCATAGAGTCAAGCCACATCCAGCA

AGCGAGGTTCGACCCGACACACGTGCTTGGGGGGCAAGTCGGGCTGGCGACGCGGAATAGGCCATTGATAGCAGAACT

CAGGTTGGGCCATTCTGTGCTGAATAGCGGTGACCGTCGTCGGTGACCATTGTCCTAATCGTCTCGCTCCATACATCC

GCCACGATGTCTCAAGAACTTCACCACCGGATTGATGCCGATGTGATCTTCCTGTCATAAACCATAGACGCGAGACGA

CTTCGGTCAATGGAAGCCTTTTTCTCAACCATCGAGAACTAGGCCGTTTGTTTGGTGGCGACCATCGCCACCAAAAAA

ACAAACGTTCGTCGTCTAATGCGCGTCTTTTTTTCCTAGAGTTCTTCTAGGAAACTAGAAAAGATGCCCCAGACTGCG

AGTCACCTTGCTTTTGAGTGCAATTCCCTAAAACCAGTACTCTAATAGTTTTTCCTAGAAGTGGATCTAGGAAAATTT

AATTTTTACTTCAAAATTTAGTTAGATTTCATATATACTCATTTGAACCAGACTGTCAATGGTTACGAATTAGTCACT

CCGTGGATAGAGTCGCTAGACAGATAAAGCAAGTAGGTATCAACGGACTGAGGGGCAGCACATCTATTGATGCTATGC

CCTCCCGAATGGTAGACCGGGGTCACGACGTTACTATGGCGCTCTGGGTGCGAGTGGCCGAGGTCTAAATAGTCGTTA

TTTGGTCGGTCGGCCTTCCCGGCTCGCGTCTTCACCAGGACGTTGAAATAGGCGGAGGTAGGTCAGATAATTAACAAC

GGCCCTTCGATCTCATTCATCAAGCGGTCAATTATCAAACGCGTTGCAACAACGGTAACGATGTCCGTAGCACCACAG

TGCGAGCAGCAAACCATACCGAAGTAAGTCGAGGCCAAGGGTTGCTAGTTCCGCTCAATGTACTAGGGGGTACAACAC

GTTTTTTCGCCAATCGAGGAAGCCAGGAGGCTAGCAACAGTCTTCATTCAACCGGCGTCACAATAGTGAGTACCAATA

CCGTCGTGACGTATTAAGAGAATGACAGTACGGTAGGCATTCTACGAAAAGACACTGACCACTCATGAGTTGGTTCAG

TAAGACTCTTATCACATACGCCGCTGGCTCAACGAGAACGGGCCGCAGTTGTGCCCTATTATGGCGCGGTGTATCGTC

TTGAAATTTTCACGAGTAGTAACCTTTTGCAAGAAGCCCCGCTTTTGAGAGTTCCTAGAATGGCGACAACTCTAGGTC

AAGCTACATTGGGTGAGCACGTGGGTTGACTAGAAGTCGTAGAAAATGAAAGTGGTCGCAAAGACCCACTCGTTTTTG

TCCTTCCGTTTTACGGCGTTTTTTCCCTTATTCCCGCTGTGCCTTTACAACTTATGAGTATGAGAAGGAAAAAGTTAT

1

79

157

235

313

391

469

547

625

703

781

859

937

1015

1093

1171

1249

1327

1405

1483

1561

1639

1717

1795

TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG

GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAA

TAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA

GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG

TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACCG

AATCGCGCGGAACTAACGACAGTCGCTCCAAGGTCGTCGAACAAAAGGTGAATGTGTTGCGGAGAGCGGGTGGGAGAC

AGCGAAAGAGCAACTACGAAACGTGGTGTGGTGGAGGTGAATTATGAAGAGGGCGCGCGATTTGAAAAGTATGTATAT

AAAAAATATATCCCGGTGTTTTATGTAGCGATAAACGAGTTTTTGATGTAAGGTATGCAGGTGTGTAAGTCTTTTGGT

TAGAAGACAAATCCAAAGTCTACTTGTGGGGATGTTCGAAGGGGAAATACTTGTATTCTATAGGTCATATCTTGTTTT

TATTGGCACAAATATAATTACATTAGCTTTTTGAGGGGGCAATAAACAGTAAACACGATGGTAATAATGGTAAAAAAA

AAAACAAGCAGTTATTTCGGATATATGTCGGCTACTCCTTGCGTCGGGCCCGAAGTCTTAGAGCCAGATATGCGAGCA

CCCGGAAGCTCACGATGAGAATGGCCAGACCATGATGAAATAACATAAGGTGGTCCCGTCGGCAAGAGACATCCACTT

AACGTATGCTTGCAATAAGTGCGAGTGAAAGGAATAGTATTCTGAGTGTCGTATTGAGTCTGAGTGAGACAGCGATAT

GATTGTTGATTAACCCTTAGCATGTCCGTGGGGTTTGAATTAACTCATAATATTAATTAGACGAAATTATTTTTAAAG

TTTTATTTTTAATAATTTGCGAGTACGCAAAGCTTCTGCATGAGCTCGGATCCAAGCTTGCATGCCTGCAGGTCGGAG

TACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTA

CTGTCCTCCGAGCGGAGACTCTAGCGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTC

AAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCTGC

AGTAAAGTGCAAGTTAAAGTGAATCAATTAAAAGTAACCAGCAACCAAGTAAATCAACTGCAACTACTGAAATCTGCC

AATAACTTCGTAAATAGTCCCAATAACAGAGTACTCGCCTATGTATAAACTTACATAAATCTTTTTATTTGTTTATCC

CCAAGGCGCGTGTAAAGGGGCTTTTCACGGTGGACTGCAGATTCTTTGGTAATAATAGTACTGTAATTGGATATTTTT

ATCCGCATAGTGCTCCGGGAAAGCAGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTCGAGGGCCT

CTGCCAGTGTCGAACAGACATTCGCCTACGGCCCTCGTCTGTTCGGGCAGTCCCGCGCAGTCGCCCACAACCGCCCAC

AGCCCCGACCGAATTGATACGCCGTAGTCTCGTCTAACATGACTCTCACGTGGTATACGCCACACTTTATGGCGTGGC

TTAGCGCGCCTTGATTGCTGTCAGCGAGGTTCCAGCAGCTTGTTTTCCACTTACACAACGCCTCTCGCCCACCCTCTG

TCGCTTTCTCGTTGATGCTTTGCACCACACCACCTCCACTTAATACTTCTCCCGCGCGCTAAACTTTTCATACATATA

TTTTTTATATAGGGCCACAAAATACATCGCTATTTGCTCAAAAACTACATTCCATACGTCCACACATTCAGAAAACCA

ATCTTCTGTTTAGGTTTCAGATGAACACCCCTACAAGCTTCCCCTTTATGAACATAAGATATCCAGTATAGAACAAAA

ATAACCGTGTTTATATTAATGTAATCGAAAAACTCCCCCGTTATTTGTCATTTGTGCTACCATTATTACCATTTTTTT

TTTTGTTCGTCAATAAAGCCTATATACAGCCGATGAGGAACGCAGCCCGGGCTTCAGAATCTCGGTCTATACGCTCGT

GGGCCTTCGAGTGCTACTCTTACCGGTCTGGTACTACTTTATTGTATTCCACCAGGGCAGCCGTTCTCTGTAGGTGAA

TTGCATACGAACGTTATTCACGCTCACTTTCCTTATCATAAGACTCACAGCATAACTCAGACTCACTCTGTCGCTATA

CTAACAACTAATTGGGAATCGTACAGGCACCCCAAACTTAATTGAGTATTATAATTAATCTGCTTTAATAAAAATTTC

AAAATAAAAATTATTAAACGCTCATGCGTTTCGAAGACGTACTCGAGCCTAGGTTCGAACGTACGGACGTCCAGCCTC

ATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCAT

GACAGGAGGCTCGCCTCTGAGATCGCTCGCGGCCTCATATTTATCTCCGCGAAGCAGATGCCTCGCTGTTAAGTTAAG

TTTGTTCGTTTCACTTGTGCAGCGATTCGCTTTCGATTCGTTTATTTGTTCGCGTCGACTTGTTCGATTTGTTAGACG

TCATTTCACGTTCAATTTCACTTAGTTAATTTTCATTGGTCGTTGGTTCATTTAGTTGACGTTGATGACTTTAGACGG

3 ' P

UAS sites

1873

1951

2029

2107

2185

2263

2341

2419

2497

2575

2653

2731

2809

2887

2965

3043

3121

3199

3277

AAGAAGTAATTATTGAATACAAGAAGAGAACTCTGAATAGGGAATTGGGGAATTCGTTAACAGATCTGACAAGTTTGT

ACAAAAAAGCAGGCTCCAGGAAA ATG ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG

GCC CTA CCG CCG CCC ACC GCA GGC ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC

CAC TGT CAG TGG AAT TCG GGC AAG AAG AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC

AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG CAG GTG CTG GAC TTT GCC CAC AAT CAA

ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG GCC GGT CTG CCC AAT GTG CAC AAG

ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT CGC GAG GCC TTC AAG GGT CTG

CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA CGG GAA CTG CAT CCG GGC

TTCTTCATTAATAACTTATGTTCTTCTCTTGAGACTTATCCCTTAACCCCTTAAGCAATTGTCTAGACTGTTCAAACA

TGTTTTTTCGTCCGAGGTCCTTT TAC TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC

CGG GAT GGC GGC GGG TGG CGT CCG TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG

GTG ACA GTC ACC TTA AGC CCG TTC TTC TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG

TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC GTC CAC GAC CTG AAA CGG GTG TTA GTT

TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC CGG CCA GAC GGG TTA CAC GTG TTC

TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA GCG CTC CGG AAG TTC CCA GAC

GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT GCC CTT GAC GTA GGC CCG

M I L L L L G V L V V L M

A L P P P T A G T T D W M Q S C G T C

H C Q W N S G K K S A D C K N K A L T

K I P Q D M S N E M Q V L D F A H N Q

I P E L R R E E F L L A G L P N V H K

I F L R N C T I Q E V H R E A F K G L

H I L I E L D L S G N R I R E L H P G

T F A G L E K L R N V I I N N N E I E

V L P N H L F V N L S F L S R I E F R

N N R L R Q V Q L H V F A G T M A L S

A I S L E Q N R L S H L H K E T F K D

L Q K L M H L S L Q G N A W N C S C E

L Q D F R D F A I S K R L Y T P P T D

C Q E P P Q L R G K L W S E V P S E N

F A C R P R I L G S V R S F I E A N H

D N I S L P C R I V G S P R P N V T W

V Y N K R P L Q Q Y D P R V R V L T S

V E Q M P E Q P S Q V L T S E L R I V

G V R A S D K G A Y T C V A D N R G G

R A E A E F Q L L V S G D Y A G A V S

A S D G M G M G A I G A P T I D P Q T

N M F L I I C L I I T T L L L L L L V

A V L T L F W Y C R R I K T Y Q K D T

T M M S G D G L I S S K M D K T H N G

S M L E G S V I M E M Q K S L L N E V

N P V E K P P R R T D I E S V D G G D

D V L E I K K T L L D D T V Y V A N H

S R D E E A V S V A M S D T T T T P R

S R H T Y V D D A Y A N S L P Y G I R

S P P S L T S P V Y T H M T P H G I Y

G T K T M T A P H N G F M T L Q H P K

S R N L A L I A T T N S S R Q H Q H H

H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q

Q Q Q Q Q H P L A T T S P F L P A P V

V Y S P A T G V V M K Q G Y M T I P R

K P R A P S W A P S T S G A A G H G S

I Q L S E F Q S P T S P N P S E T G T

A T T A E L Q A E P V Y D N L G L R T

T A G G N S T L N L T K I A G S Q G G

A G Q Q Y S M R D R P L P A T P S L T

S V S S A T N A S K I Y E P I H E L I

Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S

M D T E P L Y G V R Q Q G I T I L P G

S S I S G A G L G H A A Y L S P G S G

A A V S P S H A S S S G D S P K A A K

I P P R P P P K P K K K M S V T T T R

S G Q G S T S Q L F D D E G E D G T E

V D P A F L Y K V V V P R A R D P P V

A T M V S K G E E L F T G V V P I L V

E L D G D V N G H K F S V S G E G E G

D A T Y G K L T L K F I C T T G K L P

V P W P T L V T T L T Y G V Q C F S R

Y P D H M K Q H D F F K S A M P E G Y

V Q E R T I F F K D D G N Y K T R A E

V K F E G D T L V N R I E L K G I D F

K E D G N I L G H K L E Y N Y N S H N

V Y I M A D K Q K N G I K V N F K I R

H N I E D G S V Q L A D H Y Q Q N T P

I G D G P V L L P D N H Y L S T Q S A

L S K D P N E K R D H M V L L E F V T

A A G I T L G M D E L Y K •

EcoR1

P o l y l i n k e r

HpaI BglII attB1

signal sequence

KEK5

N FLANK

LRRs

BglII

3355

3433

3495

3552

3609

3666

3723

3780

1

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gTG ATC ATC AAC AAC AAC GAG ATC GAG

GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC ATC GAG TTC CGG

AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG GCG CTG AGC

GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC AAG GAT

CTG CAG AAG CTG ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC GAG

CTG CAG GAC TTT CGC GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT

TGC CAG GAG CCG CCA CAG CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC

TTC GCC TGC CGG CCG CGC ATT TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC

TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cAC TAG TAG TTG TTG TTG CTC TAG CTC

CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG TAG CTC AAG GCC

TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC CGC GAC TCG

CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG TTC CTA

GAC GTC TTC GAC TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG CTC

GAC GTC CTG AAA GCG CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA

ACG GTC CTC GGC GGT GTC GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG

AAG CGG ACG GCC GGC GCG TAA AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG






























































Actual sequence is GTG - should match original wt sequence

oligo238

oligo594

C FLANK

Ig

3837

3894

3951

4008

4065

4122

4179

4236

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

GAC AAT ATC TCG CTA CCC TGC CGC ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG

GTG TAC AAC AAG CGG CCA TTG CAG CAG TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC

GTG GAA CAG ATG CCG GAG CAG CCC TCC CAG GTG CTC ACC TCG GAG CTG CGC ATC GTG

GGC GTA CGG GCC TCC GAC AAG GGT GCC TAC ACC TGT GTG GCG GAT AAC CGG GGC GGA

CGG GCG GAG GCC GAG TTC CAG CTG CTC GTG AGC GGT GAC TAT GCC GGC GCG GTA TCC

GCC TCC GAT GGC ATG GGC ATG GGC GCC ATT GGG GCA CCA ACC ATT GAT CCG CAA ACG

AAC ATG TTT CTC ATC ATC TGT CTA ATC ATT ACG ACG CTG CTG CTC CTG CTG CTC GTG

GCG GTG CTG ACG CTC TTC TGG TAC TGC CGT CGC ATC AAG ACC TAT CAA AAG GAC ACC

CTG TTA TAG AGC GAT GGG ACG GCG TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC

CAC ATG TTG TTC GCC GGT AAC GTC GTC ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG

CAC CTT GTC TAC GGC CTC GTC GGG AGG GTC CAC GAG TGG AGC CTC GAC GCG TAG CAC

CCG CAT GCC CGG AGG CTG TTC CCA CGG ATG TGG ACA CAC CGC CTA TTG GCC CCG CCT

GCC CGC CTC CGG CTC AAG GTC GAC GAG CAC TCG CCA CTG ATA CGG CCG CGC CAT AGG

CGG AGG CTA CCG TAC CCG TAC CCG CGG TAA CCC CGT GGT TGG TAA CTA GGC GTT TGC

TTG TAC AAA GAG TAG TAG ACA GAT TAG TAA TGC TGC GAC GAC GAG GAC GAC GAG CAC

CGC CAC GAC TGC GAG AAG ACC ATG ACG GCA GCG TAG TTC TGG ATA GTT TTC CTG TGG






























































end Ig?

Tm

IC ∆123 Confirmed∆1235 Confirmed∆1234 Confirmed


4293

4350

4407

4464

4521

4578

4635

4692

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT TCC AAG ATG GAC AAG ACG CAC AAC GGC

TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG CAG AAG AGC CTG CTC AAC GAG GTC

AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC GAG AGC GTG GAT GGT GGC GAT

GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC GTC TAT GTG GCC AAT CAC

TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG ACG ACC ACG CCC CGA

TCT CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA TAC GGC ATC CGT

TCG CCA CCG TCG CTA ACC AGT CCG GTC TAC ACG CAT ATG ACG CCG CAC GGC ATC TAC

GGC ACC AAG ACG ATG ACG GCT CCG CAT AAC GGC TTT ATG ACG CTG CAG CAT CCC AAG

TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA AGG TTC TAC CTG TTC TGC GTG TTG CCG

AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC GTC TTC TCG GAC GAG TTG CTC CAG

TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG CTC TCG CAC CTA CCA CCG CTA

CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG CAG ATA CAC CGG TTA GTG

AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC TGC TGG TGC GGG GCT

AGA GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT ATG CCG TAG GCA

AGC GGT GGC AGC GAT TGG TCA GGC CAG ATG TGC GTA TAC TGC GGC GTG CCG TAG ATG

CCG TGG TTC TGC TAC TGC CGA GGC GTA TTG CCG AAA TAC TGC GAC GTC GTA GGG TTC






























































Good sequence for B1 & B4


∆1 ConfirmedIC1

oligo240

∆IC2 ChloeL

4749

4806

4863

4920

4977

5034

5091

5148

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

TCG CGC AAC CTG GCG CTC ATT GCC ACC ACC AAC AGC AGT CGC CAG CAC CAG CAC CAC

CAT CAG CTG CAG CAG CAG CAG CAG CAC CAC CAC CAC CAC CAG CAG CAA CAA CAA CAG

CAG CAG CAG CAG CAA CAT CCG CTG GCC ACC ACA TCG CCC TTC CTG CCC GCA CCC GTC

GTC TAT TCG CCG GCC ACG GGT GTG GTC ATG AAA CAG GGA TAT ATG ACC ATT CCG CGC

AAG CCG CGC GCT CCC AGC TGG GCG CCC AGT ACT TCC GGT GCC GCT GGC CAC GGA TCC

ATT CAG CTA AGT GAA TTC CAG AGC CCC ACA TCG CCG AAT CCC AGC GAG ACT GGC ACC

GCC ACC ACC GCG GAA CTG CAG GCG GAG CCA GTG TAC GAC AAC TTG GGA TTG CGA ACC

ACT GCC GGC GGC AAC TCC ACC CTC AAT CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC

AGC GCG TTG GAC CGC GAG TAA CGG TGG TGG TTG TCG TCA GCG GTC GTG GTC GTG GTG

GTA GTC GAC GTC GTC GTC GTC GTC GTG GTG GTG GTG GTG GTC GTC GTT GTT GTT GTC

GTC GTC GTC GTC GTT GTA GGC GAC CGG TGG TGT AGC GGG AAG GAC GGG CGT GGG CAG

CAG ATA AGC GGC CGG TGC CCA CAC CAG TAC TTT GTC CCT ATA TAC TGG TAA GGC GCG

TTC GGC GCG CGA GGG TCG ACC CGC GGG TCA TGA AGG CCA CGG CGA CCG GTG CCT AGG

TAA GTC GAT TCA CTT AAG GTC TCG GGG TGT AGC GGC TTA GGG TCG CTC TGA CCG TGG

CGG TGG TGG CGC CTT GAC GTC CGC CTC GGT CAC ATG CTG TTG AAC CCT AAC GCT TGG

TGA CGG CCG CCG TTG AGG TGG GAG TTA GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG






























































IC3 modified IC3

oligo241

∆45 Confirmed∆4 ConfirmedIC4 modified

IC4

5205

5262

5319

5376

5433

5490

5547

5604

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

GCT GGT CAG CAG TAC TCG ATG CGG GAC CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA

TCG GTG TCC TCG GCG ACC AAT GCC AGT AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT

CAG CAG CAA CAG CAG TTG CAA CAA CAA CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC

ATG GAC ACG GAA CCC CTG TAC GGA GTT CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC

TCG AGC ATT AGC GGT GCC GGA CTG GGC CAC GCC GCC TAC CTT TCA CCC GGC TCG GGT

GCC GCC GTA TCG CCA AGC CAC GCC AGC AGC AGC GGT GAC TCT CCG AAG GCC GCC AAG

ATC CCA CCA CGC CCA CCA CCG AAG CCC AAG AAG AAG ATG TCC GTG ACG ACG ACG CGC

AGC GGC CAG GGC AGC ACC AGC CAG CTC TTC GAC GAC GAG GGC GAG GAT GGC ACC GAG

CGA CCA GTC GTC ATG AGC TAC GCC CTG GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT

AGC CAC AGG AGC CGC TGG TTA CGG TCA TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA

GTC GTC GTT GTC GTC AAC GTT GTT GTT GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG

TAC CTG TGC CTT GGG GAC ATG CCT CAA GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG

AGC TCG TAA TCG CCA CGG CCT GAC CCG GTG CGG CGG ATG GAA AGT GGG CCG AGC CCA

CGG CGG CAT AGC GGT TCG GTG CGG TCG TCG TCG CCA CTG AGA GGC TTC CGG CGG TTC

TAG GGT GGT GCG GGT GGT GGC TTC GGG TTC TTC TTC TAC AGG CAC TGC TGC TGC GCG

TCG CCG GTC CCG TCG TGG TCG GTC GAG AAG CTG CTG CTC CCG CTC CTA CCG TGG CTC






























































End of Good sequence for B1 & B4

∆1235 Confirmed∆5 ConfirmedIC5

good sequence 5' to this siteIC6 (PDZ)

5661

5718

5775

5832

5889

5946

6003

6060

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

GTC GAC CCA GCT TTC TTG TAC AAA GTG GTG GTA CCG CGG GCC CGG GAT CCA CCG GTC

GCC ACC ATG GTG AGC AAG GGC GAG GAG CTG TTC ACC GGG GTG GTG CCC ATC CTG GTC

GAG CTG GAC GGC GAC GTA AAC GGC CAC AAG TTC AGC GTG TCC GGC GAG GGC GAG GGC

GAT GCC ACC TAC GGC AAG CTG ACC CTG AAG TTC ATC TGC ACC ACC GGC AAG CTG CCC

GTG CCC TGG CCC ACC CTC GTG ACC ACC CTG ACC TAC GGC GTG CAG TGC TTC AGC CGC

TAC CCC GAC CAC ATG AAG CAG CAC GAC TTC TTC AAG TCC GCC ATG CCC GAA GGC TAC

GTC CAG GAG CGC ACC ATC TTC TTC AAG GAC GAC GGC AAC TAC AAG ACC CGC GCC GAG

GTG AAG TTC GAG GGC GAC ACC CTG GTG AAC CGC ATC GAG CTG AAG GGC ATC GAC TTC

CAG CTG GGT CGA AAG AAC ATG TTT CAC CAC CAT GGC GCC CGG GCC CTA GGT GGC CAG

CGG TGG TAC CAC TCG TTC CCG CTC CTC GAC AAG TGG CCC CAC CAC GGG TAG GAC CAG

CTC GAC CTG CCG CTG CAT TTG CCG GTG TTC AAG TCG CAC AGG CCG CTC CCG CTC CCG

CTA CGG TGG ATG CCG TTC GAC TGG GAC TTC AAG TAG ACG TGG TGG CCG TTC GAC GGG

CAC GGG ACC GGG TGG GAG CAC TGG TGG GAC TGG ATG CCG CAC GTC ACG AAG TCG GCG

ATG GGG CTG GTG TAC TTC GTC GTG CTG AAG AAG TTC AGG CGG TAC GGG CTT CCG ATG

CAG GTC CTC GCG TGG TAG AAG AAG TTC CTG CTG CCG TTG ATG TTC TGG GCG CGG CTC

CAC TTC AAG CTC CCG CTG TGG GAC CAC TTG GCG TAG CTC GAC TTC CCG TAG CTG AAG






























































attL2

attB2KpnI oligo 445 T7

EGFPN1 polyl inker

EGFPN1

#54

6117

6174

6231

6288

6345

6402

6459

6516

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

AAG GAG GAC GGC AAC ATC CTG GGG CAC AAG CTG GAG TAC AAC TAC AAC AGC CAC AAC

GTC TAT ATC ATG GCC GAC AAG CAG AAG AAC GGC ATC AAG GTG AAC TTC AAG ATC CGC

CAC AAC ATC GAG GAC GGC AGC GTG CAG CTC GCC GAC CAC TAC CAG CAG AAC ACC CCC

ATC GGC GAC GGC CCC GTG CTG CTG CCC GAC AAC CAC TAC CTG AGC ACC CAG TCC GCC

CTG AGC AAA GAC CCC AAC GAG AAG CGC GAT CAC ATG GTC CTG CTG GAG TTC GTG ACC

GCC GCC GGG ATC ACT CTC GGC ATG GAC GAG CTG TAC AAG TAA AGCGGCCGCGACTCTAGAGGAT

CTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAA

TATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAAC

TGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGA

TGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTC

AGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGA

TTC CTC CTG CCG TTG TAG GAC CCC GTG TTC GAC CTC ATG TTG ATG TTG TCG GTG TTG

CAG ATA TAG TAC CGG CTG TTC GTC TTC TTG CCG TAG TTC CAC TTG AAG TTC TAG GCG

GTG TTG TAG CTC CTG CCG TCG CAC GTC GAG CGG CTG GTG ATG GTC GTC TTG TGG GGG

TAG CCG CTG CCG GGG CAC GAC GAC GGG CTG TTG GTG ATG GAC TCG TGG GTC AGG CGG

GAC TCG TTT CTG GGG TTG CTC TTC GCG CTA GTG TAC CAG GAC GAC CTC AAG CAC TGG

CGG CGG CCC TAG TGA GAG CCG TAC CTG CTC GAC ATG TTC ATT TCGCCGGCGCTGAGATCTCCTA

GAAACACTTCCTTGGAATGAAGACACCACACTGTATTAACCTGTTTGATGGATGTCTCTAAATTTCGAGATTCCATTT

ATATTTTAAAAATTCACATATTACACAATTTGATGACTAAGATTAACAAACACATAAAATCTAAGGTTGGATACCTTG

ACTACTTACCCTCGTCACCACCTTACGGAAATTACTCCTTTTGGACAAAACGAGTCTTCTTTACGGTAGATCACTACT

ACTCCGATGACGACTGAGAGTTGTAAGATGAGGAGGTTTTTTCTTCTCTTTCCATCTTCTGGGGTTCCTGAAAGGAAG

TCTTAACGATTCAAAAAACTCAGTACGACACAAATCATTATCTTGAGAACGAACGAAACGATAAATGTGGTGTTTCCT






























































Xba I SV40 Poly A

6573

6630

6687

6744

6801

6858

6922

7000

7078

7156

7234

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

1135

AAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCA

TAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTT

TAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATA

CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAA

TTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAG

CATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCGGATCCACTA

GAAGGCCTTAGTATGTATGTAAGTTAATAAAACCCTTTTTTGGAGAATGTAGATTTAAAAAAACATATTTTTTTTTTA

TTTTTTACTGCACTGGACATCATTGAACTTATCTGATCAGTTTTAAATTTACTTCGATCCAAGGGTATTTGAAGTACC

AGGTTCTTTCGATTACCTCTCACTCAAAATGACATTCCACTCAAAGTCAGCGCTGTTTGCCTCCTTCTCTGTCCACAG

AAATATCGCCGTCTCTTTCGCCGCTGCGTCCGCTATCTCTTTCGCCACCGTTTGTAGCGTTACCTAGCGTCAATGTCC

GCCTTCAGTTGCACTTTGTCAGCGGTTTCGTGACGAAGCTCCAAGCGGTTTACGCCATCAATTAAACACAAAGTGCTG

TGCCAAAACTCCTCTCGCTTCTTATTTTTGTTTGTTTTTTGAGTGATTGGGGTGGTGATTGGTTTTGGGTGGGTAAGC

AGGGGAAAGTGTGAAAAATCCCGGCAATGGGCCAAGAGGATCAGGAGCTATTAATTCGCGGAGGCAGCAAACACCCAT

CTGCCGAGCATCTGAACAATGTGAGTAGTACATGTGCATACATCTTAAGTTCACTTGATCTATAGGAACTGCGATTGC

AACATCAAATTGTCTGCGGCGTGAGAACTGCGACCCACAAAAATCCCAAACCGCAATCGCACAAACAAATAGTGACAC

GAAACAGATTATTCTGGTAGCTGTGCTCGCTATATAAGACAATTTTTAAGATCATATCATGATCAAGACATCTAAAGG

TTTTCGACGTGACGATATGTTCTTTTAATACCTTTTTATAAGACATTGGAAATATTCATCCGTATTGTCAATATTAGT

ATTGTATGACAAAAAAGAATGAGGTGTGTCCGTATCTCACAGACGATAATTATTGATACGAGTTTTTAACACATGGAA

ATCGAAAAATTAAACATTTCCCCAATTATTCCTTATAAACTACATATCACGGAACTGATCTCTAGTATTAGTCGGTAT

GGTGTAAACATCTCCAAAATGAACGAAATTTTTTGGAGGGTGTGGAGGGGGACTTGGACTTTGTATTTTACTTACGTT

AACAACAACAATTGAACAAATAACGTCGAATATTACCAATGTTTATTTCGTTATCGTAGTGTTTAAAGTGTTTATTTC

GTAAAAAAAGTGACGTAAGATCAACACCAAACAGGTTTGAGTAGTTACATAGAATAGTACAGACCTAGCCTAGGTGAT

CTTCCGGAATCATACATACATTCAATTATTTTGGGAAAAAACCTCTTACATCTAAATTTTTTTGTATAAAAAAAAAAT

AAAAAATGACGTGACCTGTAGTAACTTGAATAGACTAGTCAAAATTTAAATGAAGCTAGGTTCCCATAAACTTCATGG

TCCAAGAAAGCTAATGGAGAGTGAGTTTTACTGTAAGGTGAGTTTCAGTCGCGACAAACGGAGGAAGAGACAGGTGTC

TTTATAGCGGCAGAGAAAGCGGCGACGCAGGCGATAGAGAAAGCGGTGGCAAACATCGCAATGGATCGCAGTTACAGG

CGGAAGTCAACGTGAAACAGTCGCCAAAGCACTGCTTCGAGGTTCGCCAAATGCGGTAGTTAATTTGTGTTTCACGAC

ACGGTTTTGAGGAGAGCGAAGAATAAAAACAAACAAAAAACTCACTAACCCCACCACTAACCAAAACCCACCCATTCG

TCCCCTTTCACACTTTTTAGGGCCGTTACCCGGTTCTCCTAGTCCTCGATAATTAAGCGCCTCCGTCGTTTGTGGGTA

GACGGCTCGTAGACTTGTTACACTCATCATGTACACGTATGTAGAATTCAAGTGAACTAGATATCCTTGACGCTAACG

TTGTAGTTTAACAGACGCCGCACTCTTGACGCTGGGTGTTTTTAGGGTTTGGCGTTAGCGTGTTTGTTTATCACTGTG

CTTTGTCTAATAAGACCATCGACACGAGCGATATATTCTGTTAAAAATTCTAGTATAGTACTAGTTCTGTAGATTTCC

HpaI

B a m H I

white gene

7312

7390

7468

7546

7624

7702

7780

7858

7936

8014

8092

8170

8248

8326

8404

8482

CATTCATTTTCGACTACATTCTTTTTTACAAAAAATATAACAACCAGATATTTTAAGCTGATCCTAGATGCACAAAAA

ATAAATAAAAGTATAAACCTACTTCGTAGGATACTTCGTTTTGTTCGGGGTTAGATGAGCATAACGCTTGTAGTTGAT

ATTTGAGATCCCCTATCATTGCAGGGTGACAGCGGAGCGGCTTCGCAGAGCTGCATTAACCAGGGCTTCGGGCAGGCC

AAAAACTACGGCACGCTCCTGCCACCCAGTCCGCCGGAGGACTCCGGTTCAGGGAGCGGCCAACTAGCCGAGAACCTC

ACCTATGCCTGGCACAATATGGACATCTTTGGGGCGGTCAATCAGCCGGGCTCCGGATGGCGGCAGCTGGTCAACCGG

ACACGCGGACTATTCTGCAACGAGCGACACATACCGGCGCCCAGGAAACATTTGCTCAAGAACGGTGAGTTTCTATTC

GCAGTCGGCTGATCTGTGTGAAATCTTAATAAAGGGTCCAATTACCAATTTGAAACTCAGTTTGCGGCGTGGCCTATC

CGGGCGAACTTTTGGCCGTGATGGGCAGTTCCGGTGCCGGAAAGACGACCCTGCTGAATGCCCTTGCCTTTCGATCGC

CGCAGGGCATCCAAGTATCGCCATCCGGGATGCGACTGCTCAATGGCCAACCTGTGGACGCCAAGGAGATGCAGGCCA

GGTGCGCCTATGTCCAGCAGGATGACCTCTTTATCGGCTCCCTAACGGCCAGGGAACACCTGATTTTCCAGGCCATGG

TGCGGATGCCACGACATCTGACCTATCGGCAGCGAGTGGCCCGCGTGGATCAGGTGATCCAGGAGCTTTCGCTCAGCA

AATGTCAGCACACGATCATCGGTGTGCCCGGCAGGGTGAAAGGTCTGTCCGGCGGAGAAAGGAAGCGTCTGGCATTCG

CCTCCGAGGCACTAACCGATCCGCCGCTTCTGATCTGCGATGAGCCCACCTCCGGACTGGACTCATTTACCGCCCACA

GCGTCGTCCAGGTGCTGAAGAAGCTGTCGCAGAAGGGCAAGACCGTCATCCTGACCATTCATCAGCCGTCTTCCGAGC

TGTTTGAGCTCTTTGACAAGATCCTTCTGATGGCCGAGGGCAGGGTAGCTTTCTTGGGCACTCCCAGCGAAGCCGTCG

ACTTCTTTTCCTAGTGAGTTCGATGTGTTTATTAAGGGTATCTAGCATTACATTACATCTCAACTCCTATCCAGCGTG

GGTGCCCAGTGTCCTACCAACTACAATCCGGCGGACTTTTACGTACAGGTGTTGGCCGTTGTGCCCGGACGGGAGATC

GTAAGTAAAAGCTGATGTAAGAAAAAATGTTTTTTATATTGTTGGTCTATAAAATTCGACTAGGATCTACGTGTTTTT

TATTTATTTTCATATTTGGATGAAGCATCCTATGAAGCAAAACAAGCCCCAATCTACTCGTATTGCGAACATCAACTA

TAAACTCTAGGGGATAGTAACGTCCCACTGTCGCCTCGCCGAAGCGTCTCGACGTAATTGGTCCCGAAGCCCGTCCGG

TTTTTGATGCCGTGCGAGGACGGTGGGTCAGGCGGCCTCCTGAGGCCAAGTCCCTCGCCGGTTGATCGGCTCTTGGAG

TGGATACGGACCGTGTTATACCTGTAGAAACCCCGCCAGTTAGTCGGCCCGAGGCCTACCGCCGTCGACCAGTTGGCC

TGTGCGCCTGATAAGACGTTGCTCGCTGTGTATGGCCGCGGGTCCTTTGTAAACGAGTTCTTGCCACTCAAAGATAAG

CGTCAGCCGACTAGACACACTTTAGAATTATTTCCCAGGTTAATGGTTAAACTTTGAGTCAAACGCCGCACCGGATAG

GCCCGCTTGAAAACCGGCACTACCCGTCAAGGCCACGGCCTTTCTGCTGGGACGACTTACGGGAACGGAAAGCTAGCG

GCGTCCCGTAGGTTCATAGCGGTAGGCCCTACGCTGACGAGTTACCGGTTGGACACCTGCGGTTCCTCTACGTCCGGT

CCACGCGGATACAGGTCGTCCTACTGGAGAAATAGCCGAGGGATTGCCGGTCCCTTGTGGACTAAAAGGTCCGGTACC

ACGCCTACGGTGCTGTAGACTGGATAGCCGTCGCTCACCGGGCGCACCTAGTCCACTAGGTCCTCGAAAGCGAGTCGT

TTACAGTCGTGTGCTAGTAGCCACACGGGCCGTCCCACTTTCCAGACAGGCCGCCTCTTTCCTTCGCAGACCGTAAGC

GGAGGCTCCGTGATTGGCTAGGCGGCGAAGACTAGACGCTACTCGGGTGGAGGCCTGACCTGAGTAAATGGCGGGTGT

CGCAGCAGGTCCACGACTTCTTCGACAGCGTCTTCCCGTTCTGGCAGTAGGACTGGTAAGTAGTCGGCAGAAGGCTCG

ACAAACTCGAGAAACTGTTCTAGGAAGACTACCGGCTCCCGTCCCATCGAAAGAACCCGTGAGGGTCGCTTCGGCAGC

TGAAGAAAAGGATCACTCAAGCTACACAAATAATTCCCATAGATCGTAATGTAATGTAGAGTTGAGGATAGGTCGCAC

CCACGGGTCACAGGATGGTTGATGTTAGGCCGCCTGAAAATGCATGTCCACAACCGGCAACACGGGCCTGCCCTCTAG

8560

8638

8716

8794

8872

8950

9028

9106

9184

9262

9340

9418

9496

9574

9652

9730

9808

GAGTCCCGTGATCGGATCGCCAAGATATGCGACAATTTTGCTATTAGCAAAGTAGCCCGGGATATGGAGCAGTTGTTG

GCCACCAAAAATTTGGAGAAGCCACTGGAGCAGCCGGAGAATGGGTACACCTACAAGGCCACCTGGTTCATGCAGTTC

CGGGCGGTCCTGTGGCGATCCTGGCTGTCGGTGCTCAAGGAACCACTCCTCGTAAAAGTGCGACTTATTCAGACAACG

GTGAGTGGTTCCAGTGGAAACAAATGATATAACGCTTACAATTCTTGGAAACAAATTCGCTAGATTTTAGTTAGAATT

GCCTGATTCCACACCCTTCTTAGTTTTTTTCAATGAGATGTATAGTTTATAGTTTTGCAGAAAATAAATAAATTTCAT

TTAACTCGCGAACATGTTGAAGATATGAATATTAATGAGATGCGAGTAACATTTTAATTTGCAGATGGTTGCCATCTT

GATTGGCCTCATCTTTTTGGGCCAACAACTCACGCAAGTGGGCGTGATGAATATCAACGGAGCCATCTTCCTCTTCCT

GACCAACATGACCTTTCAAAACGTCTTTGCCACGATAAATGTAAGTCTTGTTTAGAATACATTTGCATATTAATAATT

TACTAACTTTCTAATGAATCGATTCGATTTAGGTGTTCACCTCAGAGCTGCCAGTTTTTATGAGGGAGGCCCGAAGTC

GACTTTATCGCTGTGACACATACTTTCTGGGCAAAACGATTGCCGAATTACCGCTTTTTCTCACAGTGCCACTGGTCT

TCACGGCGATTGCCTATCCGATGATCGGACTGCGGGCCGGAGTGCTGCACTTCTTCAACTGCCTGGCGCTGGTCACTC

TGGTGGCCAATGTGTCAACGTCCTTCGGATATCTAATATCCTGCGCCAGCTCCTCGACCTCGATGGCGCTGTCTGTGG

GTCCGCCGGTTATCATACCATTCCTGCTCTTTGGCGGCTTCTTCTTGAACTCGGGCTCGGTGCCAGTATACCTCAAAT

GGTTGTCGTACCTCTCATGGTTCCGTTACGCCAACGAGGGTCTGCTGATTAACCAATGGGCGGACGTGGAGCCGGGCG

AAATTAGCTGCACATCGTCGAACACCACGTGCCCCAGTTCGGGCAAGGTCATCCTGGAGACGCTTAACTTCTCCGCCG

CCGATCTGCCGCTGGACTACGTGGGTCTGGCCATTCTCATCGTGAGCTTCCGGGTGCTCGCATATCTGGCTCTAAGAC

TTCGGGCCCGACGCAAGGAGTAGCCGACATATATCCGAAATAACTGCTTGTTTTTTTTTTTACCATTATTACCATCGT

CTCAGGGCACTAGCCTAGCGGTTCTATACGCTGTTAAAACGATAATCGTTTCATCGGGCCCTATACCTCGTCAACAAC

CGGTGGTTTTTAAACCTCTTCGGTGACCTCGTCGGCCTCTTACCCATGTGGATGTTCCGGTGGACCAAGTACGTCAAG

GCCCGCCAGGACACCGCTAGGACCGACAGCCACGAGTTCCTTGGTGAGGAGCATTTTCACGCTGAATAAGTCTGTTGC

CACTCACCAAGGTCACCTTTGTTTACTATATTGCGAATGTTAAGAACCTTTGTTTAAGCGATCTAAAATCAATCTTAA

CGGACTAAGGTGTGGGAAGAATCAAAAAAAGTTACTCTACATATCAAATATCAAAACGTCTTTTATTTATTTAAAGTA

AATTGAGCGCTTGTACAACTTCTATACTTATAATTACTCTACGCTCATTGTAAAATTAAACGTCTACCAACGGTAGAA

CTAACCGGAGTAGAAAAACCCGGTTGTTGAGTGCGTTCACCCGCACTACTTATAGTTGCCTCGGTAGAAGGAGAAGGA

CTGGTTGTACTGGAAAGTTTTGCAGAAACGGTGCTATTTACATTCAGAACAAATCTTATGTAAACGTATAATTATTAA

ATGATTGAAAGATTACTTAGCTAAGCTAAATCCACAAGTGGAGTCTCGACGGTCAAAAATACTCCCTCCGGGCTTCAG

CTGAAATAGCGACACTGTGTATGAAAGACCCGTTTTGCTAACGGCTTAATGGCGAAAAAGAGTGTCACGGTGACCAGA

AGTGCCGCTAACGGATAGGCTACTAGCCTGACGCCCGGCCTCACGACGTGAAGAAGTTGACGGACCGCGACCAGTGAG

ACCACCGGTTACACAGTTGCAGGAAGCCTATAGATTATAGGACGCGGTCGAGGAGCTGGAGCTACCGCGACAGACACC

CAGGCGGCCAATAGTATGGTAAGGACGAGAAACCGCCGAAGAAGAACTTGAGCCCGAGCCACGGTCATATGGAGTTTA

CCAACAGCATGGAGAGTACCAAGGCAATGCGGTTGCTCCCAGACGACTAATTGGTTACCCGCCTGCACCTCGGCCCGC

TTTAATCGACGTGTAGCAGCTTGTGGTGCACGGGGTCAAGCCCGTTCCAGTAGGACCTCTGCGAATTGAAGAGGCGGC

GGCTAGACGGCGACCTGATGCACCCAGACCGGTAAGAGTAGCACTCGAAGGCCCACGAGCGTATAGACCGAGATTCTG

AAGCCCGGGCTGCGTTCCTCATCGGCTGTATATAGGCTTTATTGACGAACAAAAAAAAAAATGGTAATAATGGTAGCA

9886

9964

10042

10120

10198

10276

10354

10432

10510

10588

10666

10744

10822

10900

10978

11056

11134

GTTTACTGTTTATTGCCCCCTCAAAAAGCTAATGTAATTATATTTGTGCCAATAAAAACAAGATATGACCTATAGAAT

ACAAGTATTTCCCCTTCGAACATCCCCACAAGTAGACTTTGGATTTGTCTTCTAACCAAAAGACTTACACACCTGCAT

ACCTTACATCAAAAACTCGTTTATCGCTACATAAAACACCGGGATATATTTTTTATATACATACTTTTCAAATCGCGC

GCCCTCTTCATAATTCACCTCCACCACACCACGTTTCGTAGTTGCTCTTTCGCTGTCTCCCACCCGCTCTCCGCAACA

CATTCACCTTTTGTTCGACGACCTTGGAGCGACTGTCGTTAGTTCCGCGCGATTCGGTTCGCTCAAATGGTTCCGAGT

GGTTCATTTCGTCTCAATAGAAATTAGTAATAAATATTTGTATGTACAATTTATTTGCTCCAATATATTTGTATATAT

TTCCCTCACAGCTATATTTATTCTAATTTAATATTATGACTTTTTAAGGTAATTTTTTGTGACCTGTTCGGAGTGATT

AGCGTTACAATTTGAACTGAAAGTGACATCCAGTGTTTGTTCCTTGTGTAGATGCATCTCAAAAAAATGGTGGGCATA

ATAGTGTTGTTTATATATATCAAAAATAACAACTATAATAATAAGAATACATTTAATTTAGAAAATGCTTGGATTTCA

CTGGAACTAGAATTAATTCGGCTGCTGCTCTAAACGACGCATTTCGTACTCCAAAGTACGAATTTTTTCCCTCAAGCT

CTTATTTTCATTAAACAATGAACAGGACCTAACGCACAGTCACGTTATTGTTTACATAAATGATTTTTTTTACTATTC

AAACTTACTCTGTTTGTGTACTCCCACTGGTATAGCCTTCTTTTATCTTTTCTGGTTCAGGCTCTATCACTTTACTAG

GTACGGCATCTGCGTTGAGTCGCCTCCTTTTAAATGTCTGACCTTTTGCAGGTGCAGCCTTCCACTGCGAATCATTAA

AGTGGGTATCACAAATTTGGGAGTTTTCACCAAGGCTGCACCCAAGGCTCTGCTCCCACAATTTTCTCTTAATAGCAC

ACTTCGGCACGTGAATTAATTTTACTCCAGTCACAGCTTTGCAGCAAAATTTGCAATATTTCATTTTTTTTTATTCCA

CGTAAGGGTTAATGTTTTCAAAAAAAAATTCGTCCGCACACAACCTTTCCTCTCAACAAGCAAACGTGCACTGAATTT

CAAATGACAAATAACGGGGGAGTTTTTCGATTACATTAATATAAACACGGTTATTTTTGTTCTATACTGGATATCTTA

TGTTCATAAAGGGGAAGCTTGTAGGGGTGTTCATCTGAAACCTAAACAGAAGATTGGTTTTCTGAATGTGTGGACGTA

TGGAATGTAGTTTTTGAGCAAATAGCGATGTATTTTGTGGCCCTATATAAAAAATATATGTATGAAAAGTTTAGCGCG

CGGGAGAAGTATTAAGTGGAGGTGGTGTGGTGCAAAGCATCAACGAGAAAGCGACAGAGGGTGGGCGAGAGGCGTTGT

GTAAGTGGAAAACAAGCTGCTGGAACCTCGCTGACAGCAATCAAGGCGCGCTAAGCCAAGCGAGTTTACCAAGGCTCA

CCAAGTAAAGCAGAGTTATCTTTAATCATTATTTATAAACATACATGTTAAATAAACGAGGTTATATAAACATATATA

AAGGGAGTGTCGATATAAATAAGATTAAATTATAATACTGAAAAATTCCATTAAAAAACACTGGACAAGCCTCACTAA

TCGCAATGTTAAACTTGACTTTCACTGTAGGTCACAAACAAGGAACACATCTACGTAGAGTTTTTTTACCACCCGTAT

TATCACAACAAATATATATAGTTTTTATTGTTGATATTATTATTCTTATGTAAATTAAATCTTTTACGAACCTAAAGT

GACCTTGATCTTAATTAAGCCGACGACGAGATTTGCTGCGTAAAGCATGAGGTTTCATGCTTAAAAAAGGGAGTTCGA

GAATAAAAGTAATTTGTTACTTGTCCTGGATTGCGTGTCAGTGCAATAACAAATGTATTTACTAAAAAAAATGATAAG

TTTGAATGAGACAAACACATGAGGGTGACCATATCGGAAGAAAATAGAAAAGACCAAGTCCGAGATAGTGAAATGATC

CATGCCGTAGACGCAACTCAGCGGAGGAAAATTTACAGACTGGAAAACGTCCACGTCGGAAGGTGACGCTTAGTAATT

TCACCCATAGTGTTTAAACCCTCAAAAGTGGTTCCGACGTGGGTTCCGAGACGAGGGTGTTAAAAGAGAATTATCGTG

TGAAGCCGTGCACTTAATTAAAATGAGGTCAGTGTCGAAACGTCGTTTTAAACGTTATAAAGTAAAAAAAAATAAGGT

GCATTCCCAATTACAAAAGTTTTTTTTTAAGCAGGCGTGTGTTGGAAAGGAGAGTTGTTCGTTTGCACGTGACTTAAA

11212

11290

11368

11446

11524

11602

11680

11758

11836

11914

11992

12070

12148

12226

12304

12382

AAGTGTATACTTCGGTAAGCTTCGGCTATCGACGGGACCACCTTATGTTATTTCATCATGTTCACATATGAAGCCATTCGAAGCCGATAGCTGCCCTGGTGGAATACAATAAAGTAGTAC

5' P

5' P

12460

GGCCAGACCCACGTAGTCCAGCGGCAGATCGGCGGCGGAGAAGTTAAGCGTCTCCAGGATGACCTTGCCCGAACTGGG

GCACGTGGTGTTCGACGATGTGCAGCTAATTTCGCCCGGCTCCACGTCCGCCCATTGGTTAATCAGCAGACCCTCGTT

GGCGTAACGGAACCATGAGAGGTACGACAACCATTTGAGGTATACTGGCACCGAGCCCGAGTTCAAGAAGAAGGCGTT

TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT

ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT

GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT

TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA

GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG

CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT

GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT

TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC

TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA

TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGA

GGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG

GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT

AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG

CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC

ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTG

CAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT

GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC

ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAG

AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC

AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA

CCGGTCTGGGTGCATCAGGTCGCCGTCTAGCCGCCGCCTCTTCAATTCGCAGAGGTCCTACTGGAACGGGCTTGACCC

CGTGCACCACAAGCTGCTACACGTCGATTAAAGCGGGCCGAGGTGCAGGCGGGTAACCAATTAGTCGTCTGGGAGCAA

CCGCATTGCCTTGGTACTCTCCATGCTGTTGGTAAACTCCATATGACCGTGGCTCGGGCTCAAGTTCTTCTTCCGCAA

AAAGGTATCCGAGGCGGGGGGACTGCTCGTAGTGTTTTTAGCTGCGAGTTCAGTCTCCACCGCTTTGGGCTGTCCTGA

TATTTCTATGGTCCGCAAAGGGGGACCTTCGAGGGAGCACGCGAGAGGACAAGGCTGGGACGGCGAATGGCCTATGGA

CAGGCGGAAAGAGGGAAGCCCTTCGCACCGCGAAAGAGTTACGAGTGCGACATCCATAGAGTCAAGCCACATCCAGCA

AGCGAGGTTCGACCCGACACACGTGCTTGGGGGGCAAGTCGGGCTGGCGACGCGGAATAGGCCATTGATAGCAGAACT

CAGGTTGGGCCATTCTGTGCTGAATAGCGGTGACCGTCGTCGGTGACCATTGTCCTAATCGTCTCGCTCCATACATCC

GCCACGATGTCTCAAGAACTTCACCACCGGATTGATGCCGATGTGATCTTCCTGTCATAAACCATAGACGCGAGACGA

CTTCGGTCAATGGAAGCCTTTTTCTCAACCATCGAGAACTAGGCCGTTTGTTTGGTGGCGACCATCGCCACCAAAAAA

ACAAACGTTCGTCGTCTAATGCGCGTCTTTTTTTCCTAGAGTTCTTCTAGGAAACTAGAAAAGATGCCCCAGACTGCG

AGTCACCTTGCTTTTGAGTGCAATTCCCTAAAACCAGTACTCTAATAGTTTTTCCTAGAAGTGGATCTAGGAAAATTT

AATTTTTACTTCAAAATTTAGTTAGATTTCATATATACTCATTTGAACCAGACTGTCAATGGTTACGAATTAGTCACT

CCGTGGATAGAGTCGCTAGACAGATAAAGCAAGTAGGTATCAACGGACTGAGGGGCAGCACATCTATTGATGCTATGC

CCTCCCGAATGGTAGACCGGGGTCACGACGTTACTATGGCGCTCTGGGTGCGAGTGGCCGAGGTCTAAATAGTCGTTA

TTTGGTCGGTCGGCCTTCCCGGCTCGCGTCTTCACCAGGACGTTGAAATAGGCGGAGGTAGGTCAGATAATTAACAAC

GGCCCTTCGATCTCATTCATCAAGCGGTCAATTATCAAACGCGTTGCAACAACGGTAACGATGTCCGTAGCACCACAG

TGCGAGCAGCAAACCATACCGAAGTAAGTCGAGGCCAAGGGTTGCTAGTTCCGCTCAATGTACTAGGGGGTACAACAC

GTTTTTTCGCCAATCGAGGAAGCCAGGAGGCTAGCAACAGTCTTCATTCAACCGGCGTCACAATAGTGAGTACCAATA

CCGTCGTGACGTATTAAGAGAATGACAGTACGGTAGGCATTCTACGAAAAGACACTGACCACTCATGAGTTGGTTCAG

TAAGACTCTTATCACATACGCCGCTGGCTCAACGAGAACGGGCCGCAGTTGTGCCCTATTATGGCGCGGTGTATCGTC

TTGAAATTTTCACGAGTAGTAACCTTTTGCAAGAAGCCCCGCTTTTGAGAGTTCCTAGAATGGCGACAACTCTAGGTC

AAGCTACATTGGGTGAGCACGTGGGTTGACTAGAAGTCGTAGAAAATGAAAGTGGTCGCAAAGACCCACTCGTTTTTG

TCCTTCCGTTTTACGGCGTTTTTTCCCTTATTCCCGCTGTGCCTTTACAACTTATGAGTATGAGAAGGAAAAAGTTAT

1

79

157

235

313

391

469

547

625

703

781

859

937

1015

1093

1171

1249

1327

1405

1483

1561

1639

1717

1795

TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG

GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAA

TAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA

GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG

TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACCG

AATCGCGCGGAACTAACGACAGTCGCTCCAAGGTCGTCGAACAAAAGGTGAATGTGTTGCGGAGAGCGGGTGGGAGAC

AGCGAAAGAGCAACTACGAAACGTGGTGTGGTGGAGGTGAATTATGAAGAGGGCGCGCGATTTGAAAAGTATGTATAT

AAAAAATATATCCCGGTGTTTTATGTAGCGATAAACGAGTTTTTGATGTAAGGTATGCAGGTGTGTAAGTCTTTTGGT

TAGAAGACAAATCCAAAGTCTACTTGTGGGGATGTTCGAAGGGGAAATACTTGTATTCTATAGGTCATATCTTGTTTT

TATTGGCACAAATATAATTACATTAGCTTTTTGAGGGGGCAATAAACAGTAAACACGATGGTAATAATGGTAAAAAAA

AAAACAAGCAGTTATTTCGGATATATGTCGGCTACTCCTTGCGTCGGGCCCGAAGTCTTAGAGCCAGATATGCGAGCA

CCCGGAAGCTCACGATGAGAATGGCCAGACCATGATGAAATAACATAAGGTGGTCCCGTCGGCAAGAGACATCCACTT

AACGTATGCTTGCAATAAGTGCGAGTGAAAGGAATAGTATTCTGAGTGTCGTATTGAGTCTGAGTGAGACAGCGATAT

GATTGTTGATTAACCCTTAGCATGTCCGTGGGGTTTGAATTAACTCATAATATTAATTAGACGAAATTATTTTTAAAG

TTTTATTTTTAATAATTTGCGAGTACGCAAAGCTTCTGCATGAGCTCGGATCCAAGCTTGCATGCCTGCAGGTCGGAG

TACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTA

CTGTCCTCCGAGCGGAGACTCTAGCGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTC

AAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCTGC

AGTAAAGTGCAAGTTAAAGTGAATCAATTAAAAGTAACCAGCAACCAAGTAAATCAACTGCAACTACTGAAATCTGCC

GTACTACTTTATTGTATTCCACCAGGGCAGCCGTTCTCTGTAGGTGAA

TTGCATACGAACGTTATTCACGCTCACTTTCCTTATCATAAGACTCACAGCATAACTCAGACTCACTCTGTCGCTATA

CTAACAACTAATTGGGAATCGTACAGGCACCCCAAACTTAATTGAGTATTATAATTAATCTGCTTTAATAAAAATTTC

AAAATAAAAATTATTAAACGCTCATGCGTTTCGAAGACGTACTCGAGCCTAGGTTCGAACGTACGGACGTCCAGCCTC

ATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCAT

GACAGGAGGCTCGCCTCTGAGATCGCTCGCGGCCTCATATTTATCTCCGCGAAGCAGATGCCTCGCTGTTAAGTTAAG

TTTGTTCGTTTCACTTGTGCAGCGATTCGCTTTCGATTCGTTTATTTGTTCGCGTCGACTTGTTCGATTTGTTAGACG

TCATTTCACGTTCAATTTCACTTAGTTAATTTTCATTGGTCGTTGGTTCATTTAGTTGACGTTGATGACTTTAGACGG

AATAACTTCGTAAATAGTCCCAATAACAGAGTACTCGCCTATGTATAAACTTACATAAATCTTTTTATTTGTTTATCC

CCAAGGCGCGTGTAAAGGGGCTTTTCACGGTGGACTGCAGATTCTTTGGTAATAATAGTACTGTAATTGGATATTTTT

ATCCGCATAGTGCTCCGGGAAAGCAGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTCGAGGGCCT

CTGCCAGTGTCGAACAGACATTCGCCTACGGCCCTCGTCTGTTCGGGCAGTCCCGCGCAGTCGCCCACAACCGCCCAC

AGCCCCGACCGAATTGATACGCCGTAGTCTCGTCTAACATGACTCTCACGTGGTATACGCCACACTTTATGGCGTGGC

TTAGCGCGCCTTGATTGCTGTCAGCGAGGTTCCAGCAGCTTGTTTTCCACTTACACAACGCCTCTCGCCCACCCTCTG

TCGCTTTCTCGTTGATGCTTTGCACCACACCACCTCCACTTAATACTTCTCCCGCGCGCTAAACTTTTCATACATATA

TTTTTTATATAGGGCCACAAAATACATCGCTATTTGCTCAAAAACTACATTCCATACGTCCACACATTCAGAAAACCA

ATCTTCTGTTTAGGTTTCAGATGAACACCCCTACAAGCTTCCCCTTTATGAACATAAGATATCCAGTATAGAACAAAA

ATAACCGTGTTTATATTAATGTAATCGAAAAACTCCCCCGTTATTTGTCATTTGTGCTACCATTATTACCATTTTTTT

TTTTGTTCGTCAATAAAGCCTATATACAGCCGATGAGGAACGCAGCCCGGGCTTCAGAATCTCGGTCTATACGCTCGT

GGGCCTTCGAGTGCTACTCTTACCGGTCTG

3 ' P

UAS sites

1873

1951

2029

2107

2185

2263

2341

2419

2497

2575

2653

2731

2809

2887

2965

3043

3121

3199

3277

AAGAAGTAATTATTGAATACAAGAAGAGAACTCTGAATAGGGAATTGGGGAATTCGTTAACAGATCTGACAAGTTTGT

ACAAAAAAGCAGGCTCCAGGAAA ATG ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG

GCC CTA CCG CCG CCC ACC GCA GGC ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC

CAC TGT CAG TGG AAT TCG GGC AAG AAG AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC

AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG CAG GTG CTG GAC TTT GCC CAC AAT CAA

ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG GCC GGT CTG CCC AAT GTG CAC AAG

ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT CGC GAG GCC TTC AAG GGT CTG

CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA CGG GAA CTG CAT CCG GGC

ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC ATG ATC ATC AAC AAC AAC GAG ATC GAG

GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC ATC GAG TTC CGG

AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG GCG CTG AGC

TTCTTCATTAATAACTTATGTTCTTCTCTTGAGACTTATCCCTTAACCCCTTAAGCAATTGTCTAGACTGTTCAAACA

TGTTTTTTCGTCCGAGGTCCTTT TAC TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC

CGG GAT GGC GGC GGG TGG CGT CCG TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG

GTG ACA GTC ACC TTA AGC CCG TTC TTC TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG

TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC GTC CAC GAC CTG AAA CGG GTG TTA GTT

TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC CGG CCA GAC GGG TTA CAC GTG TTC

TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA GCG CTC CGG AAG TTC CCA GAC

GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT GCC CTT GAC GTA GGC CCG

TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG TAC TAG TAG TTG TTG TTG CTC TAG CTC

CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG TAG CTC AAG GCC

TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC CGC GAC TCG








T F A G L E K L R N M I I N N N E I E





















S R H T Y V D D A Y A N S L P P D L L

A F P A R V P P T S P S M Q S S Q S N

I P D Q V I Y G I R S P P S L T S P V

Y T H M T P H G I Y G T K T M T A P H

N G F M T L Q H P K S R N L A L I A T

T N S S R Q H Q H H H Q L Q Q Q Q Q H

H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A

T T S P F L P A P V V Y S P A T G V E

P V Y D N L G L R T T A G G N S T L N

L T K I A G S Q G G A G Q Q Y S M R D

R P L P A T P S L T S V S S A T N A S

K I Y E P I H E L I Q Q Q Q Q L Q Q Q

Q Q Q Q Q Q R L G S M D T E P L Y G V

R Q Q G I T I L P G S S I S G A G L G

H A A Y L S P G S G A A V S P S H A S

S S G D S P K A A K I P P R P P P K P

K K K M S V T T T R S G Q G S T S Q L

F D D E G E D G T E V D P A F L Y K V

V V P R A R D P P V A T M V S K G E E

L F T G V V P I L V E L D G D V N G H

K F S V S G E G E G D A T Y G K L T L

K F I C T T G K L P V P W P T L V T T

L T Y G V Q C F S R Y P D H M K Q H D

F F K S A M P E G Y V Q E R T I F F K

D D G N Y K T R A E V K F E G D T L V

N R I E L K G I D F K E D G N I L G H

K L E Y N Y N S H N V Y I M A D K Q K

N G I K V N F K I R H N I E D G S V Q

L A D H Y Q Q N T P I G D G P V L L P

D N H Y L S T Q S A L S K D P N E K R

D H M V L L E F V T A A G I T L G M D

E L Y K •

EcoR1

P o l y l i n k e r

HpaI BglII attB1

K5signal sequence

KEK5

Start of Good Sequence

N FLANK

LRRs

BglII

originally C

oligo238

3355

3433

3495

3552

3609

3666

3723

3780

3837

3894

3951

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC AAG GAT

CTG CAG AAG CTG ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC GAG

CTG CAG GAC TTT CGC GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT

TGC CAG GAG CCG CCA CAG CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC

TTC GCC TGC CGG CCG CGC ATT TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC

GAC AAT ATC TCG CTA CCC TGC CGC ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG

GTG TAC AAC AAG CGG CCA TTG CAG CAG TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC

GTG GAA CAG ATG CCG GAG CAG CCC TCC CAG GTG CTC ACC TCG GAG CTG CGC ATC GTG

GGC GTA CGG GCC TCC GAC AAG GGT GCC TAC ACC TGT GTG GCG GAT AAC CGG GGC GGA

CGG GCG GAG GCC GAG TTC CAG CTG CTC GTG AGC GGT GAC TAT GCC GGC GCG GTA TCC

GCC TCC GAT GGC ATG GGC ATG GGC GCC ATT GGG GCA CCA ACC ATT GAT CCG CAA ACG

AAC ATG TTT CTC ATC ATC TGT CTA ATC ATT ACG ACG CTG CTG CTC CTG CTG CTC GTG

CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG TTC CTA

GAC GTC TTC GAC TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG CTC

GAC GTC CTG AAA GCG CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA

ACG GTC CTC GGC GGT GTC GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG

AAG CGG ACG GCC GGC GCG TAA AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG

CTG TTA TAG AGC GAT GGG ACG GCG TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC

CAC ATG TTG TTC GCC GGT AAC GTC GTC ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG

CAC CTT GTC TAC GGC CTC GTC GGG AGG GTC CAC GAG TGG AGC CTC GAC GCG TAG CAC

CCG CAT GCC CGG AGG CTG TTC CCA CGG ATG TGG ACA CAC CGC CTA TTG GCC CCG CCT

GCC CGC CTC CGG CTC AAG GTC GAC GAG CAC TCG CCA CTG ATA CGG CCG CGC CAT AGG

CGG AGG CTA CCG TAC CCG TAC CCG CGG TAA CCC CGT GGT TGG TAA CTA GGC GTT TGC

TTG TAC AAA GAG TAG TAG ACA GAT TAG TAA TGC TGC GAC GAC GAG GAC GAC GAG CAC




























































E L Y K •

oligo594

C FLANK

Ig

end Ig?

Tm

4008

4065

4122

4179

4236

4293

4350

4407

4464

4521

4578

4635

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

GCG GTG CTG ACG CTC TTC TGG TAC TGC CGT CGC ATC AAG ACC TAT CAA AAG GAC ACC

ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT TCC AAG ATG GAC AAG ACG CAC AAC GGC

TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG CAG AAG AGC CTG CTC AAC GAG GTC

AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC GAG AGC GTG GAT GGT GGC GAT

GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC GTC TAT GTG GCC AAT CAC

TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG ACG ACC ACG CCC CGA

TCT CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA CCG GAT CTG CTG

GCC TTT CCC GCT CGC GTG CCG CCC ACC TCG CCC TCG ATG CAA TCG TCG CAG TCG AAC

ATA CCC GAC CAG GTG ATC TAC GGC ATC CGT TCG CCA CCG TCG CTA ACC AGT CCG GTC

TAC ACG CAT ATG ACG CCG CAC GGC ATC TAC GGC ACC AAG ACG ATG ACG GCT CCG CAT

AAC GGC TTT ATG ACG CTG CAG CAT CCC AAG TCG CGC AAC CTG GCG CTC ATT GCC ACC

CGC CAC GAC TGC GAG AAG ACC ATG ACG GCA GCG TAG TTC TGG ATA GTT TTC CTG TGG

TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA AGG TTC TAC CTG TTC TGC GTG TTG CCG

AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC GTC TTC TCG GAC GAG TTG CTC CAG

TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG CTC TCG CAC CTA CCA CCG CTA

CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG CAG ATA CAC CGG TTA GTG

AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC TGC TGG TGC GGG GCT

AGA GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT GGC CTA GAC GAC

CGG AAA GGG CGA GCG CAC GGC GGG TGG AGC GGG AGC TAC GTT AGC AGC GTC AGC TTG

TAT GGG CTG GTC CAC TAG ATG CCG TAG GCA AGC GGT GGC AGC GAT TGG TCA GGC CAG

ATG TGC GTA TAC TGC GGC GTG CCG TAG ATG CCG TGG TTC TGC TAC TGC CGA GGC GTA

TTG CCG AAA TAC TGC GAC GTC GTA GGG TTC AGC GCG TTG GAC CGC GAG TAA CGG TGG




























































E L Y K •

IC oligo745 apK5_seq_int_5'


∆1 ConfirmedIC1

oligo240

∆234 ConfirmedIC2 modified

IC2

4692

4749

4806

4863

4920

4977

5034

5091

5148

5205

5262

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

ACC AAC AGC AGT CGC CAG CAC CAG CAC CAC CAT CAG CTG CAG CAG CAG CAG CAG CAC

CAC CAC CAC CAC CAG CAG CAA CAA CAA CAG CAG CAG CAG CAG CAA CAT CCG CTG GCC

ACC ACA TCG CCC TTC CTG CCC GCA CCC GTC GTC TAT TCG CCG GCC ACG GGT GTG GAG

CCA GTG TAC GAC AAC TTG GGA TTG CGA ACC ACT GCC GGC GGC AAC TCC ACC CTC AAT

CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC GCT GGT CAG CAG TAC TCG ATG CGG GAC

CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA TCG GTG TCC TCG GCG ACC AAT GCC AGT

AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT CAG CAG CAA CAG CAG TTG CAA CAA CAA

CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC ATG GAC ACG GAA CCC CTG TAC GGA GTT

CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC TCG AGC ATT AGC GGT GCC GGA CTG GGC

CAC GCC GCC TAC CTT TCA CCC GGC TCG GGT GCC GCC GTA TCG CCA AGC CAC GCC AGC

AGC AGC GGT GAC TCT CCG AAG GCC GCC AAG ATC CCA CCA CGC CCA CCA CCG AAG CCC

AAG AAG AAG ATG TCC GTG ACG ACG ACG CGC AGC GGC CAG GGC AGC ACC AGC CAG CTC

TGG TTG TCG TCA GCG GTC GTG GTC GTG GTG GTA GTC GAC GTC GTC GTC GTC GTC GTG

GTG GTG GTG GTG GTC GTC GTT GTT GTT GTC GTC GTC GTC GTC GTT GTA GGC GAC CGG

TGG TGT AGC GGG AAG GAC GGG CGT GGG CAG CAG ATA AGC GGC CGG TGC CCA CAC CTC

GGT CAC ATG CTG TTG AAC CCT AAC GCT TGG TGA CGG CCG CCG TTG AGG TGG GAG TTA

GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG CGA CCA GTC GTC ATG AGC TAC GCC CTG

GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT AGC CAC AGG AGC CGC TGG TTA CGG TCA

TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA GTC GTC GTT GTC GTC AAC GTT GTT GTT

GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG TAC CTG TGC CTT GGG GAC ATG CCT CAA

GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG AGC TCG TAA TCG CCA CGG CCT GAC CCG

GTG CGG CGG ATG GAA AGT GGG CCG AGC CCA CGG CGG CAT AGC GGT TCG GTG CGG TCG

TCG TCG CCA CTG AGA GGC TTC CGG CGG TTC TAG GGT GGT GCG GGT GGT GGC TTC GGG

TTC TTC TTC TAC AGG CAC TGC TGC TGC GCG TCG CCG GTC CCG TCG TGG TCG GTC GAG




























































E L Y K •

∆ IC3CLIC4

IC5

5319

5376

5433

5490

5547

5604

5661

5718

5775

5832

5889

5946

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

TTC GAC GAC GAG GGC GAG GAT GGC ACC GAG GTC GAC CCA GCT TTC TTG TAC AAA GTG

GTG GTA CCG CGG GCC CGG GAT CCA CCG GTC GCC ACC ATG GTG AGC AAG GGC GAG GAG

CTG TTC ACC GGG GTG GTG CCC ATC CTG GTC GAG CTG GAC GGC GAC GTA AAC GGC CAC

AAG TTC AGC GTG TCC GGC GAG GGC GAG GGC GAT GCC ACC TAC GGC AAG CTG ACC CTG

AAG TTC ATC TGC ACC ACC GGC AAG CTG CCC GTG CCC TGG CCC ACC CTC GTG ACC ACC

CTG ACC TAC GGC GTG CAG TGC TTC AGC CGC TAC CCC GAC CAC ATG AAG CAG CAC GAC

TTC TTC AAG TCC GCC ATG CCC GAA GGC TAC GTC CAG GAG CGC ACC ATC TTC TTC AAG

GAC GAC GGC AAC TAC AAG ACC CGC GCC GAG GTG AAG TTC GAG GGC GAC ACC CTG GTG

AAC CGC ATC GAG CTG AAG GGC ATC GAC TTC AAG GAG GAC GGC AAC ATC CTG GGG CAC

AAG CTG GAG TAC AAC TAC AAC AGC CAC AAC GTC TAT ATC ATG GCC GAC AAG CAG AAG

AAC GGC ATC AAG GTG AAC TTC AAG ATC CGC CAC AAC ATC GAG GAC GGC AGC GTG CAG

CTC GCC GAC CAC TAC CAG CAG AAC ACC CCC ATC GGC GAC GGC CCC GTG CTG CTG CCC

AAG CTG CTG CTC CCG CTC CTA CCG TGG CTC CAG CTG GGT CGA AAG AAC ATG TTT CAC

CAC CAT GGC GCC CGG GCC CTA GGT GGC CAG CGG TGG TAC CAC TCG TTC CCG CTC CTC

GAC AAG TGG CCC CAC CAC GGG TAG GAC CAG CTC GAC CTG CCG CTG CAT TTG CCG GTG

TTC AAG TCG CAC AGG CCG CTC CCG CTC CCG CTA CGG TGG ATG CCG TTC GAC TGG GAC

TTC AAG TAG ACG TGG TGG CCG TTC GAC GGG CAC GGG ACC GGG TGG GAG CAC TGG TGG

GAC TGG ATG CCG CAC GTC ACG AAG TCG GCG ATG GGG CTG GTG TAC TTC GTC GTG CTG

AAG AAG TTC AGG CGG TAC GGG CTT CCG ATG CAG GTC CTC GCG TGG TAG AAG AAG TTC

CTG CTG CCG TTG ATG TTC TGG GCG CGG CTC CAC TTC AAG CTC CCG CTG TGG GAC CAC

TTG GCG TAG CTC GAC TTC CCG TAG CTG AAG TTC CTC CTG CCG TTG TAG GAC CCC GTG

TTC GAC CTC ATG TTG ATG TTG TCG GTG TTG CAG ATA TAG TAC CGG CTG TTC GTC TTC

TTG CCG TAG TTC CAC TTG AAG TTC TAG GCG GTG TTG TAG CTC CTG CCG TCG CAC GTC

GAG CGG CTG GTG ATG GTC GTC TTG TGG GGG TAG CCG CTG CCG GGG CAC GAC GAC GGG




























































E L Y K •

End of Good SequenceIC6 (PDZ)

attB2

KpnI oligo 445 T7

EGFPN1 polyl inker

XmaI

EGFPN1

#54

6003

6060

6117

6174

6231

6288

6345

6402

6459

6516

6573

6630

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

GAC AAC CAC TAC CTG AGC ACC CAG TCC GCC CTG AGC AAA GAC CCC AAC GAG AAG CGC

GAT CAC ATG GTC CTG CTG GAG TTC GTG ACC GCC GCC GGG ATC ACT CTC GGC ATG GAC

GAG CTG TAC AAG TAA AGCGGCCGCGACTCTAGAGGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATA

ATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACT

GATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGA

GGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCC

AAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAG

TAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAA

ATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAG

AGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATA

TTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACC

TCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATG

GTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA

AACTCATCAATGTATCTTATCATGTCTGGATCGGATCCACTAGAAGGCCTTAGTATGTATGTAAGTTAATAAAACCCT

TTTTTGGAGAATGTAGATTTAAAAAAACATATTTTTTTTTTATTTTTTACTGCACTGGACATCATTGAACTTATCTGA

CTG TTG GTG ATG GAC TCG TGG GTC AGG CGG GAC TCG TTT CTG GGG TTG CTC TTC GCG

CTA GTG TAC CAG GAC GAC CTC AAG CAC TGG CGG CGG CCC TAG TGA GAG CCG TAC CTG

CTC GAC ATG TTC ATT TCGCCGGCGCTGAGATCTCCTAGAAACACTTCCTTGGAATGAAGACACCACACTGTAT

TAACCTGTTTGATGGATGTCTCTAAATTTCGAGATTCCATTTATATTTTAAAAATTCACATATTACACAATTTGATGA

CTAAGATTAACAAACACATAAAATCTAAGGTTGGATACCTTGACTACTTACCCTCGTCACCACCTTACGGAAATTACT

CCTTTTGGACAAAACGAGTCTTCTTTACGGTAGATCACTACTACTCCGATGACGACTGAGAGTTGTAAGATGAGGAGG

TTTTTTCTTCTCTTTCCATCTTCTGGGGTTCCTGAAAGGAAGTCTTAACGATTCAAAAAACTCAGTACGACACAAATC

ATTATCTTGAGAACGAACGAAACGATAAATGTGGTGTTTCCTTTTTCGACGTGACGATATGTTCTTTTAATACCTTTT

TATAAGACATTGGAAATATTCATCCGTATTGTCAATATTAGTATTGTATGACAAAAAAGAATGAGGTGTGTCCGTATC

TCACAGACGATAATTATTGATACGAGTTTTTAACACATGGAAATCGAAAAATTAAACATTTCCCCAATTATTCCTTAT

AAACTACATATCACGGAACTGATCTCTAGTATTAGTCGGTATGGTGTAAACATCTCCAAAATGAACGAAATTTTTTGG

AGGGTGTGGAGGGGGACTTGGACTTTGTATTTTACTTACGTTAACAACAACAATTGAACAAATAACGTCGAATATTAC

CAATGTTTATTTCGTTATCGTAGTGTTTAAAGTGTTTATTTCGTAAAAAAAGTGACGTAAGATCAACACCAAACAGGT

TTGAGTAGTTACATAGAATAGTACAGACCTAGCCTAGGTGATCTTCCGGAATCATACATACATTCAATTATTTTGGGA

AAAAACCTCTTACATCTAAATTTTTTTGTATAAAAAAAAAATAAAAAATGACGTGACCTGTAGTAACTTGAATAGACT




























































E L Y K •

Xba I SV40 Poly A

HpaI

B a m H I

white gene

6687

6744

6801

6874

6952

7030

7108

7186

7264

7342

7420

7498

7576

7654

7732

1

14

33

52

71

90

109

128

147

166

185

204

223

242

261

280

299

318

337

356

375

394

413

432

451

470

489

508

527

546

565

584

603

622

641

660

679

698

717

736

755

774

793

812

831

850

869

888

907

926

945

964

983

1002

1021

1040

1059

1078

1097

1116

TCAGTTTTAAATTTACTTCGATCCAAGGGTATTTGAAGTACCAGGTTCTTTCGATTACCTCTCACTCAAAATGACATT

CCACTCAAAGTCAGCGCTGTTTGCCTCCTTCTCTGTCCACAGAAATATCGCCGTCTCTTTCGCCGCTGCGTCCGCTAT

CTCTTTCGCCACCGTTTGTAGCGTTACCTAGCGTCAATGTCCGCCTTCAGTTGCACTTTGTCAGCGGTTTCGTGACGA

AGCTCCAAGCGGTTTACGCCATCAATTAAACACAAAGTGCTGTGCCAAAACTCCTCTCGCTTCTTATTTTTGTTTGTT

TTTTGAGTGATTGGGGTGGTGATTGGTTTTGGGTGGGTAAGCAGGGGAAAGTGTGAAAAATCCCGGCAATGGGCCAAG

AGGATCAGGAGCTATTAATTCGCGGAGGCAGCAAACACCCATCTGCCGAGCATCTGAACAATGTGAGTAGTACATGTG

CATACATCTTAAGTTCACTTGATCTATAGGAACTGCGATTGCAACATCAAATTGTCTGCGGCGTGAGAACTGCGACCC

ACAAAAATCCCAAACCGCAATCGCACAAACAAATAGTGACACGAAACAGATTATTCTGGTAGCTGTGCTCGCTATATA

AGACAATTTTTAAGATCATATCATGATCAAGACATCTAAAGGCATTCATTTTCGACTACATTCTTTTTTACAAAAAAT

ATAACAACCAGATATTTTAAGCTGATCCTAGATGCACAAAAAATAAATAAAAGTATAAACCTACTTCGTAGGATACTT

CGTTTTGTTCGGGGTTAGATGAGCATAACGCTTGTAGTTGATATTTGAGATCCCCTATCATTGCAGGGTGACAGCGGA

GCGGCTTCGCAGAGCTGCATTAACCAGGGCTTCGGGCAGGCCAAAAACTACGGCACGCTCCTGCCACCCAGTCCGCCG

GAGGACTCCGGTTCAGGGAGCGGCCAACTAGCCGAGAACCTCACCTATGCCTGGCACAATATGGACATCTTTGGGGCG

GTCAATCAGCCGGGCTCCGGATGGCGGCAGCTGGTCAACCGGACACGCGGACTATTCTGCAACGAGCGACACATACCG

GCGCCCAGGAAACATTTGCTCAAGAACGGTGAGTTTCTATTCGCAGTCGGCTGATCTGTGTGAAATCTTAATAAAGGG

TCCAATTACCAATTTGAAACTCAGTTTGCGGCGTGGCCTATCCGGGCGAACTTTTGGCCGTGATGGGCAGTTCCGGTG

CCGGAAAGACGACCCTGCTGAATGCCCTTGCCTTTCGATCGCCGCAGGGCATCCAAGTATCGCCATCCGGGATGCGAC

AGTCAAAATTTAAATGAAGCTAGGTTCCCATAAACTTCATGGTCCAAGAAAGCTAATGGAGAGTGAGTTTTACTGTAA

GGTGAGTTTCAGTCGCGACAAACGGAGGAAGAGACAGGTGTCTTTATAGCGGCAGAGAAAGCGGCGACGCAGGCGATA

GAGAAAGCGGTGGCAAACATCGCAATGGATCGCAGTTACAGGCGGAAGTCAACGTGAAACAGTCGCCAAAGCACTGCT

TCGAGGTTCGCCAAATGCGGTAGTTAATTTGTGTTTCACGACACGGTTTTGAGGAGAGCGAAGAATAAAAACAAACAA

AAAACTCACTAACCCCACCACTAACCAAAACCCACCCATTCGTCCCCTTTCACACTTTTTAGGGCCGTTACCCGGTTC

TCCTAGTCCTCGATAATTAAGCGCCTCCGTCGTTTGTGGGTAGACGGCTCGTAGACTTGTTACACTCATCATGTACAC

GTATGTAGAATTCAAGTGAACTAGATATCCTTGACGCTAACGTTGTAGTTTAACAGACGCCGCACTCTTGACGCTGGG

TGTTTTTAGGGTTTGGCGTTAGCGTGTTTGTTTATCACTGTGCTTTGTCTAATAAGACCATCGACACGAGCGATATAT

TCTGTTAAAAATTCTAGTATAGTACTAGTTCTGTAGATTTCCGTAAGTAAAAGCTGATGTAAGAAAAAATGTTTTTTA

TATTGTTGGTCTATAAAATTCGACTAGGATCTACGTGTTTTTTATTTATTTTCATATTTGGATGAAGCATCCTATGAA

GCAAAACAAGCCCCAATCTACTCGTATTGCGAACATCAACTATAAACTCTAGGGGATAGTAACGTCCCACTGTCGCCT

CGCCGAAGCGTCTCGACGTAATTGGTCCCGAAGCCCGTCCGGTTTTTGATGCCGTGCGAGGACGGTGGGTCAGGCGGC

CTCCTGAGGCCAAGTCCCTCGCCGGTTGATCGGCTCTTGGAGTGGATACGGACCGTGTTATACCTGTAGAAACCCCGC

CAGTTAGTCGGCCCGAGGCCTACCGCCGTCGACCAGTTGGCCTGTGCGCCTGATAAGACGTTGCTCGCTGTGTATGGC

CGCGGGTCCTTTGTAAACGAGTTCTTGCCACTCAAAGATAAGCGTCAGCCGACTAGACACACTTTAGAATTATTTCCC

AGGTTAATGGTTAAACTTTGAGTCAAACGCCGCACCGGATAGGCCCGCTTGAAAACCGGCACTACCCGTCAAGGCCAC

GGCCTTTCTGCTGGGACGACTTACGGGAACGGAAAGCTAGCGGCGTCCCGTAGGTTCATAGCGGTAGGCCCTACGCTG

7810

7888

7966

8044

8122

8200

8278

8356

8434

8512

8590

8668

8746

8824

8902

8980

9058

TGCTCAATGGCCAACCTGTGGACGCCAAGGAGATGCAGGCCAGGTGCGCCTATGTCCAGCAGGATGACCTCTTTATCG

GCTCCCTAACGGCCAGGGAACACCTGATTTTCCAGGCCATGGTGCGGATGCCACGACATCTGACCTATCGGCAGCGAG

TGGCCCGCGTGGATCAGGTGATCCAGGAGCTTTCGCTCAGCAAATGTCAGCACACGATCATCGGTGTGCCCGGCAGGG

TGAAAGGTCTGTCCGGCGGAGAAAGGAAGCGTCTGGCATTCGCCTCCGAGGCACTAACCGATCCGCCGCTTCTGATCT

GCGATGAGCCCACCTCCGGACTGGACTCATTTACCGCCCACAGCGTCGTCCAGGTGCTGAAGAAGCTGTCGCAGAAGG

GCAAGACCGTCATCCTGACCATTCATCAGCCGTCTTCCGAGCTGTTTGAGCTCTTTGACAAGATCCTTCTGATGGCCG

AGGGCAGGGTAGCTTTCTTGGGCACTCCCAGCGAAGCCGTCGACTTCTTTTCCTAGTGAGTTCGATGTGTTTATTAAG

GGTATCTAGCATTACATTACATCTCAACTCCTATCCAGCGTGGGTGCCCAGTGTCCTACCAACTACAATCCGGCGGAC

TTTTACGTACAGGTGTTGGCCGTTGTGCCCGGACGGGAGATCGAGTCCCGTGATCGGATCGCCAAGATATGCGACAAT

TTTGCTATTAGCAAAGTAGCCCGGGATATGGAGCAGTTGTTGGCCACCAAAAATTTGGAGAAGCCACTGGAGCAGCCG

GAGAATGGGTACACCTACAAGGCCACCTGGTTCATGCAGTTCCGGGCGGTCCTGTGGCGATCCTGGCTGTCGGTGCTC

AAGGAACCACTCCTCGTAAAAGTGCGACTTATTCAGACAACGGTGAGTGGTTCCAGTGGAAACAAATGATATAACGCT

TACAATTCTTGGAAACAAATTCGCTAGATTTTAGTTAGAATTGCCTGATTCCACACCCTTCTTAGTTTTTTTCAATGA

GATGTATAGTTTATAGTTTTGCAGAAAATAAATAAATTTCATTTAACTCGCGAACATGTTGAAGATATGAATATTAAT

GAGATGCGAGTAACATTTTAATTTGCAGATGGTTGCCATCTTGATTGGCCTCATCTTTTTGGGCCAACAACTCACGCA

AGTGGGCGTGATGAATATCAACGGAGCCATCTTCCTCTTCCTGACCAACATGACCTTTCAAAACGTCTTTGCCACGAT

AAATGTAAGTCTTGTTTAGAATACATTTGCATATTAATAATTTACTAACTTTCTAATGAATCGATTCGATTTAGGTGT

ACGAGTTACCGGTTGGACACCTGCGGTTCCTCTACGTCCGGTCCACGCGGATACAGGTCGTCCTACTGGAGAAATAGC

CGAGGGATTGCCGGTCCCTTGTGGACTAAAAGGTCCGGTACCACGCCTACGGTGCTGTAGACTGGATAGCCGTCGCTC

ACCGGGCGCACCTAGTCCACTAGGTCCTCGAAAGCGAGTCGTTTACAGTCGTGTGCTAGTAGCCACACGGGCCGTCCC

ACTTTCCAGACAGGCCGCCTCTTTCCTTCGCAGACCGTAAGCGGAGGCTCCGTGATTGGCTAGGCGGCGAAGACTAGA

CGCTACTCGGGTGGAGGCCTGACCTGAGTAAATGGCGGGTGTCGCAGCAGGTCCACGACTTCTTCGACAGCGTCTTCC

CGTTCTGGCAGTAGGACTGGTAAGTAGTCGGCAGAAGGCTCGACAAACTCGAGAAACTGTTCTAGGAAGACTACCGGC

TCCCGTCCCATCGAAAGAACCCGTGAGGGTCGCTTCGGCAGCTGAAGAAAAGGATCACTCAAGCTACACAAATAATTC

CCATAGATCGTAATGTAATGTAGAGTTGAGGATAGGTCGCACCCACGGGTCACAGGATGGTTGATGTTAGGCCGCCTG

AAAATGCATGTCCACAACCGGCAACACGGGCCTGCCCTCTAGCTCAGGGCACTAGCCTAGCGGTTCTATACGCTGTTA

AAACGATAATCGTTTCATCGGGCCCTATACCTCGTCAACAACCGGTGGTTTTTAAACCTCTTCGGTGACCTCGTCGGC

CTCTTACCCATGTGGATGTTCCGGTGGACCAAGTACGTCAAGGCCCGCCAGGACACCGCTAGGACCGACAGCCACGAG

TTCCTTGGTGAGGAGCATTTTCACGCTGAATAAGTCTGTTGCCACTCACCAAGGTCACCTTTGTTTACTATATTGCGA

ATGTTAAGAACCTTTGTTTAAGCGATCTAAAATCAATCTTAACGGACTAAGGTGTGGGAAGAATCAAAAAAAGTTACT

CTACATATCAAATATCAAAACGTCTTTTATTTATTTAAAGTAAATTGAGCGCTTGTACAACTTCTATACTTATAATTA

CTCTACGCTCATTGTAAAATTAAACGTCTACCAACGGTAGAACTAACCGGAGTAGAAAAACCCGGTTGTTGAGTGCGT

TCACCCGCACTACTTATAGTTGCCTCGGTAGAAGGAGAAGGACTGGTTGTACTGGAAAGTTTTGCAGAAACGGTGCTA

TTTACATTCAGAACAAATCTTATGTAAACGTATAATTATTAAATGATTGAAAGATTACTTAGCTAAGCTAAATCCACA

XmaI

9136

9214

9292

9370

9448

9526

9604

9682

9760

9838

9916

9994

10072

10150

10228

10306

10384

TCACCTCAGAGCTGCCAGTTTTTATGAGGGAGGCCCGAAGTCGACTTTATCGCTGTGACACATACTTTCTGGGCAAAA

CGATTGCCGAATTACCGCTTTTTCTCACAGTGCCACTGGTCTTCACGGCGATTGCCTATCCGATGATCGGACTGCGGG

CCGGAGTGCTGCACTTCTTCAACTGCCTGGCGCTGGTCACTCTGGTGGCCAATGTGTCAACGTCCTTCGGATATCTAA

TATCCTGCGCCAGCTCCTCGACCTCGATGGCGCTGTCTGTGGGTCCGCCGGTTATCATACCATTCCTGCTCTTTGGCG

GCTTCTTCTTGAACTCGGGCTCGGTGCCAGTATACCTCAAATGGTTGTCGTACCTCTCATGGTTCCGTTACGCCAACG

AGGGTCTGCTGATTAACCAATGGGCGGACGTGGAGCCGGGCGAAATTAGCTGCACATCGTCGAACACCACGTGCCCCA

GTTCGGGCAAGGTCATCCTGGAGACGCTTAACTTCTCCGCCGCCGATCTGCCGCTGGACTACGTGGGTCTGGCCATTC

TCATCGTGAGCTTCCGGGTGCTCGCATATCTGGCTCTAAGACTTCGGGCCCGACGCAAGGAGTAGCCGACATATATCC

GAAATAACTGCTTGTTTTTTTTTTTACCATTATTACCATCGTGTTTACTGTTTATTGCCCCCTCAAAAAGCTAATGTA

ATTATATTTGTGCCAATAAAAACAAGATATGACCTATAGAATACAAGTATTTCCCCTTCGAACATCCCCACAAGTAGA

CTTTGGATTTGTCTTCTAACCAAAAGACTTACACACCTGCATACCTTACATCAAAAACTCGTTTATCGCTACATAAAA

CACCGGGATATATTTTTTATATACATACTTTTCAAATCGCGCGCCCTCTTCATAATTCACCTCCACCACACCACGTTT

CGTAGTTGCTCTTTCGCTGTCTCCCACCCGCTCTCCGCAACACATTCACCTTTTGTTCGACGACCTTGGAGCGACTGT

CGTTAGTTCCGCGCGATTCGGTTCGCTCAAATGGTTCCGAGTGGTTCATTTCGTCTCAATAGAAATTAGTAATAAATA

TTTGTATGTACAATTTATTTGCTCCAATATATTTGTATATATTTCCCTCACAGCTATATTTATTCTAATTTAATATTA

TGACTTTTTAAGGTAATTTTTTGTGACCTGTTCGGAGTGATTAGCGTTACAATTTGAACTGAAAGTGACATCCAGTGT

TTGTTCCTTGTGTAGATGCATCTCAAAAAAATGGTGGGCATAATAGTGTTGTTTATATATATCAAAAATAACAACTAT

AGTGGAGTCTCGACGGTCAAAAATACTCCCTCCGGGCTTCAGCTGAAATAGCGACACTGTGTATGAAAGACCCGTTTT

GCTAACGGCTTAATGGCGAAAAAGAGTGTCACGGTGACCAGAAGTGCCGCTAACGGATAGGCTACTAGCCTGACGCCC

GGCCTCACGACGTGAAGAAGTTGACGGACCGCGACCAGTGAGACCACCGGTTACACAGTTGCAGGAAGCCTATAGATT

ATAGGACGCGGTCGAGGAGCTGGAGCTACCGCGACAGACACCCAGGCGGCCAATAGTATGGTAAGGACGAGAAACCGC

CGAAGAAGAACTTGAGCCCGAGCCACGGTCATATGGAGTTTACCAACAGCATGGAGAGTACCAAGGCAATGCGGTTGC

TCCCAGACGACTAATTGGTTACCCGCCTGCACCTCGGCCCGCTTTAATCGACGTGTAGCAGCTTGTGGTGCACGGGGT

CAAGCCCGTTCCAGTAGGACCTCTGCGAATTGAAGAGGCGGCGGCTAGACGGCGACCTGATGCACCCAGACCGGTAAG

AGTAGCACTCGAAGGCCCACGAGCGTATAGACCGAGATTCTGAAGCCCGGGCTGCGTTCCTCATCGGCTGTATATAGG

CTTTATTGACGAACAAAAAAAAAAATGGTAATAATGGTAGCACAAATGACAAATAACGGGGGAGTTTTTCGATTACAT

TAATATAAACACGGTTATTTTTGTTCTATACTGGATATCTTATGTTCATAAAGGGGAAGCTTGTAGGGGTGTTCATCT

GAAACCTAAACAGAAGATTGGTTTTCTGAATGTGTGGACGTATGGAATGTAGTTTTTGAGCAAATAGCGATGTATTTT

GTGGCCCTATATAAAAAATATATGTATGAAAAGTTTAGCGCGCGGGAGAAGTATTAAGTGGAGGTGGTGTGGTGCAAA

GCATCAACGAGAAAGCGACAGAGGGTGGGCGAGAGGCGTTGTGTAAGTGGAAAACAAGCTGCTGGAACCTCGCTGACA

GCAATCAAGGCGCGCTAAGCCAAGCGAGTTTACCAAGGCTCACCAAGTAAAGCAGAGTTATCTTTAATCATTATTTAT

AAACATACATGTTAAATAAACGAGGTTATATAAACATATATAAAGGGAGTGTCGATATAAATAAGATTAAATTATAAT

ACTGAAAAATTCCATTAAAAAACACTGGACAAGCCTCACTAATCGCAATGTTAAACTTGACTTTCACTGTAGGTCACA

AACAAGGAACACATCTACGTAGAGTTTTTTTACCACCCGTATTATCACAACAAATATATATAGTTTTTATTGTTGATA

10462

10540

10618

10696

10774

10852

10930

11008

11086

11164

11242

11320

11398

11476

11554

11632

11710

AATAATAAGAATACATTTAATTTAGAAAATGCTTGGATTTCACTGGAACTAGAATTAATTCGGCTGCTGCTCTAAACG

ACGCATTTCGTACTCCAAAGTACGAATTTTTTCCCTCAAGCTCTTATTTTCATTAAACAATGAACAGGACCTAACGCA

CAGTCACGTTATTGTTTACATAAATGATTTTTTTTACTATTCAAACTTACTCTGTTTGTGTACTCCCACTGGTATAGC

CTTCTTTTATCTTTTCTGGTTCAGGCTCTATCACTTTACTAGGTACGGCATCTGCGTTGAGTCGCCTCCTTTTAAATG

TCTGACCTTTTGCAGGTGCAGCCTTCCACTGCGAATCATTAAAGTGGGTATCACAAATTTGGGAGTTTTCACCAAGGC

TGCACCCAAGGCTCTGCTCCCACAATTTTCTCTTAATAGCACACTTCGGCACGTGAATTAATTTTACTCCAGTCACAG

CTTTGCAGCAAAATTTGCAATATTTCATTTTTTTTTATTCCACGTAAGGGTTAATGTTTTCAAAAAAAAATTCGTCCG

CACACAACCTTTCCTCTCAACAAGCAAACGTGCACTGAATTTAAGTGTATACTTCGGTAAGCTTCGGCTATCGACGGG

ACCACCTTATGTTATTTCATCATG

TTATTATTCTTATGTAAATTAAATCTTTTACGAACCTAAAGTGACCTTGATCTTAATTAAGCCGACGACGAGATTTGC

TGCGTAAAGCATGAGGTTTCATGCTTAAAAAAGGGAGTTCGAGAATAAAAGTAATTTGTTACTTGTCCTGGATTGCGT

GTCAGTGCAATAACAAATGTATTTACTAAAAAAAATGATAAGTTTGAATGAGACAAACACATGAGGGTGACCATATCG

GAAGAAAATAGAAAAGACCAAGTCCGAGATAGTGAAATGATCCATGCCGTAGACGCAACTCAGCGGAGGAAAATTTAC

AGACTGGAAAACGTCCACGTCGGAAGGTGACGCTTAGTAATTTCACCCATAGTGTTTAAACCCTCAAAAGTGGTTCCG

ACGTGGGTTCCGAGACGAGGGTGTTAAAAGAGAATTATCGTGTGAAGCCGTGCACTTAATTAAAATGAGGTCAGTGTC

GAAACGTCGTTTTAAACGTTATAAAGTAAAAAAAAATAAGGTGCATTCCCAATTACAAAAGTTTTTTTTTAAGCAGGC

GTGTGTTGGAAAGGAGAGTTGTTCGTTTGCACGTGACTTAAATTCACATATGAAGCCATTCGAAGCCGATAGCTGCCC

TGGTGGAATACAATAAAGTAGTAC

5' P

5' P

5' P

11788

11866

11944

12022

12100

12178

12256

12334

12412

lajeunesse mqp 4-25dr

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