Articleshttps://doi.org/10.1038/s41587-020-0613-1

Inducible de novo expression of neoantigens in tumor cells and miceMartina Damo   1,4, Brittany Fitzgerald1,4, Yisi Lu   2, Mursal Nader1, Ivana William1, Julie F. Cheung   1, Kelli A. Connolly1, Gena G. Foster   1, Elliot Akama-Garren2, Da-Yae Lee2, Greg P. Chang2, Vasilena Gocheva2, Leah M. Schmidt2, Alice Boileve2, Josephine H. Wilson1, Can Cui   1, Isabel Monroy1, Prashanth Gokare1, Peter Cabeceiras2, Tyler Jacks   2,3,4 ✉ and Nikhil S. Joshi   1,2,4 ✉

1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA. 2Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. 3Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA. 4These authors contributed equally: Martina Damo, Brittany Fitzgerald, Tyler Jacks, Nikhil S. Joshi. ✉e-mail: tjacks@mit.edu; nikhil.joshi@yale.edu

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Supplementary Information

Supplementary Notes

Development and testing of NM constructs:

Neoantigen Module Version 1 (NM.1): We designed a proof-of-concept construct, in

which the GP33-80 region of LCMV was fused to the C-terminus of eGFP (NM.1;

Extended Data Figure 2a). To design the DNA sequence (in silico) for our initial

construct (see Extended Data Figure 1b for details), we started with the fusion of

eGFP to GP33-80 and then introduced a consensus splice donor (SD) sequence

AAG|GTGAGT 5’ to the DNA encoding the K amino acid residues highlighted above

in GP33-41 and GP66-77 (AAG encodes for K). Note, see NM.7 for explanation as to

why GP33-41 is used in methods versus GP33-43 in the main text. Just 3’ to the DNA

encoding the A and G amino acid residues in GP33-41 and GP66-77 above

(respectively), we inserted a strong splice acceptor (SA) sequence from human

adenovirus 5. We also inserted the intronic sequences from the chicken beta-actin

(cβA) gene (cβA intron or cβAi; contained in the pCAG promoter) between the splice

donor and acceptor sequences. This generated a DNA construct that was

eGFP:GP33:SD:cβAi:SA:GP34-GP68:SD:cβAi:SA:GP69-80, where the SA:GP34-

GP68:SD portion encoded exon 2. We then replaced this exon 2 sequence with its

reverse and complement (generating eGFP:GP33:SD:cβAi:SD:GP68-

GP34:SA:cβAi:SA:GP69-80), because we reasoned in this configuration exons 1 and

3 would splice directly. Splice sites were confirmed using the GENESCAN web server

at MIT (http://hollywood.mit.edu/GENSCAN.html). Finally, to ensure no spurious

transcription of exon 2 could occur, we inserted an inverted SV40 polyadenylation

sequence (iSV40pA) between the 3’ FRT-F3:FRT-WT sequences.

We then synthesized the backbone DNA for the NM.1 construct containing the WT

and F3 FRT sites in opposite orientations and multiple restriction sites to facilitate the

cloning of the subsequent parts in the pIDTBlue vector (Integrated DNA

Technologies). The vector was synthesized in two halves due to the repetitiveness of

the sequence. These halves were jointly cloned into the pBluescript II SK- vector

(Agilent Technologies). This construct had the orientation: GP33:SD:intron1:FRT-

F3:FRT-WT:SD:GP68-GP34:SA:FRT-F3:iSV40pA:FRT-WT:intron2:SA:GP69-80.

PCR amplification was used to clone GFP with a splice donor into the construct

upstream of the GP33. This construct was then cloned into the expression vector

pcDNA3 (Thermo) for testing and validation. Note, the DNA sequences for all

constructs are provided as a Supplementary Sequences document.

We transiently transfected 293T cells with the NM.1 construct alone or co-

transfected the construct with a pcDNA3 plasmid containing FLPo recombinase. Flow

cytometric (FACS) analysis showed the construct alone was fluorescent when the

neoantigen was in the OFF state (no FLPo), but when recombination was induced, we

also noted a loss of fluorescence intensity in the cells (Extended Data Figure 2b). To

determine if the protein of the correct size was produced, protein from transfected

293T cells was analyzed by western blotting with an anti-GFP antibody (clone D5.1,

Cell Signaling). This corresponded with an increase in protein size from 27 to 32 kDa

by western blotting (Extended Data Figure 2c). We also confirmed that the correctly

spliced products were generated by generating cDNA from 293T cells transfected with

the NM.1 construct with and without FLPo. cDNA was amplified using PrimeSTAR HS

DNA Polymerase (Takara Bio Inc., cat. R040A) and the primers GFP F AgeI 72 and

GP80 R XhoI 64 (Supplementary Table 2). This PCR fragment was cloned into the

pCR-Blunt TOPO kit (ThermoFisher Scientific, cat. K280002) and DNA from 7-10

clones from before and after FLPo-mediated recombination was prepared by miniprep

(Qiagen, cat. 27106) and sequenced by Sanger sequencing with the primer GFP int F

Age 66. All of the clones had the correct sequences (data not shown).

Version 2 (NM.2): Generated to test if shorter intron sequences would function, to

reduce the chances of spurious DNA recombination in cloning for future versions.

Version 3 (NM.3): Linking neoantigen expression to fluorescence. This approach was

inspired by the biofluorescence complementation (BiFC) assay46. Yellow fluorescent

protein (YFP) and GFP have beta-barrel structures consisting of 11 beta-strands that

are connected by loops, and, for BiFC, YFP or GFP is split between amino acid

residues 155 and 156 (beta-strands 7 and 8) to generate two halves. We inserted the

spliced neoantigen construct from NM.2 between amino acid residues 155 and 156 of

YFP (NM.3 - in frame; Extended Data Figures 3a-b). In the OFF-state, YFP would

also contain amino acids 33 and 69-80 from GP within the loop connecting beta-

strands 7 and 8 of YFP (Extended Data Figure 3b).

To generate a construct that would become fluorescent after FLPo induction, we

next engineered a frame shift such that YFP molecule would require exon 2 to maintain

the correct reading frame (i.e., direct splicing of exons 1 and 3 would result in a splicing

event that would alter the reading frame; Extended Data Figure 3c). This was

achieved in silico by adding an A residue into the splice donor sequence at the 3’ end

of exon 1 and deleting a G from the splice acceptor sequence at the 5’ end of exon 2.

Next, we engineered the 5’ end of exon 3 to contain a TTC -> TTT mutation that was

silent when exon 2 spliced to exon 3 (ON state) but created a premature truncation

when exon 1 spliced to exon 3. This would ensure that the OFF state NM.3 produced

only the N-terminal half of YFP.

We synthesized the NM.3 construct with the frameshift, transfected the variant into

293T cells and analyzed their fluorescence by FACS. As a control, we mutated the

NM.3 construct to remove A463, such that the splicing of exon 1 to exon 3 would occur

in frame. As expected, without the frameshift the construct was fluorescent in the OFF

state (Extended Data Figure 3d). Introduction of the frameshift into the NM.3

construct eliminated the fluorescence of YFP in the OFF state, and resulted in

production of only the N-terminus of YFP by Western (Extended Data Figure 2c).

However, the construct was not fluorescent when co-transfected with plasmid

containing FLPo (Extended Data Figure 3d), despite the production of a correctly

sized protein (Extended Data Figure 2c).

Versions 4-6 (NM.4-NM.6): To address the lack of fluorescence, we attempted a

number of modifications to the construct including the addition of linkers between the

N- and C-terminal halves of YFP and GP33-80 (NM.4) and incorporating GFP instead

of YFP to increase the sensitivity of detection by FACS (NM.5). Yet, these did not

increase the fluorescence of the constructs in the ON state, despite Western and

immunofluorescence (IF) data showing the constructs were producing the correct

proteins (Extended Data Figures 2b-c and 3e).

We noted that by IF the construct appeared to become less “sharp” after FLPo and

we reasoned this could indicate a protein folding error. Further investigation of the

GP33-80 sequence revealed that it contained a hydrophobic region between GP42-

GP59, that was strongly predicted to be a transmembrane domain and could disrupt

the folding of GFP (Extended Data Figure 3f,47). To address whether this

hydrophobic region was preventing fluorescence, we removed residues in the next

version to leave just GP33-41 and GP60-80, replacing the removed sequence with a

FLAG tag sequence and thus eliminating the predicted transmembrane domain

(NM.6). The resulting construct was indeed fluorescent in the ON state (not shown).

Thus, we had developed an inversion-mediated fluorescent neoantigen that was

regulated by FLPo. However, we then realized that GP33-41 has low affinity for H2-

Db (5429 nM IC50) compared to GP33-43, which we addressed in NM.7.

Version 7 (NM.7): In the unlikely event there was transcription of Exon 2 (in the

opposite direction), there was a concern that a peptide could be produced that could

mimic GP33-41 and cause tolerance. Note, there is an upstream SV40 poly A

sequence in the inverted direction to minimize this risk. We analyzed the potential

peptide products from this construct and realized that, due to the structure of the splice

acceptor, the DNA sequence encodes GP34-41 and that the amino acid proceeding

this was encoded by nCA, which could encode either an Ala, Ser, Thr, or Pro residue

(Extended Data Figure 4a). We calculated the predicted binding of each peptide

(GP34-41 or GP33-41 with K33A, K33S, K33T, or K33P mutations, Extended Data

Figure 4b) and found that K33A, K33S, and K33T bound as strongly or more strongly

to H2-Db as the wt GP33-41 peptide. We also confirmed that K33A, K33S, and K33T

bind to H2-Db using RMA-S cells, and that K33A and K33S could activate GP33-

specific P14 TCR Tg CD8 T cells (Extended Data Figure 4c and data not shown). By

contrast GP34-41 and K33P did not bind to H2-Db or stimulate T cell activation. GP34-

41 did bind to H2-Kb as previously reported48. We engineered the splice acceptor in

Exon 2 so that it encoded K33P in the OFF state. In addition, we became aware that

while the GP33 epitope in LCMV specific tetramers contains the “GP33-41” peptide,

this peptide contains a C to M mutation in GP41 that leads to an increased affinity for

Db (from 5429 to 27 nM IC50,49). In LCMV, the presented GP33 epitope is likely GP33-

43, which has a more physiologically relevant IC50 of 395 nM (IC50)48. Consequently,

in NM.7, we added amino acids GP42-43 (Extended Data Figure 2a – NM.7). Thus,

NM.7 has two key differences from NM.6: alteration of the intron/exon junction and the

addition of residues GP42-43. NM.7 serves as the final version of the NM in NINJA.

Development and testing of RM constructs:

Development of FLPoER251: We generated a FLPoER fusion by fusing amino acids

282-596 of the human estrogen receptor (hER) containing the T2 mutations (G400V,

M543A, and L544A;50) to the C-terminus of FLPo but identified that this had relatively

weak activity in reporter cells upon 4-hydroxytamoxifen treatment (4-OHT; Extended

Data Figure 5a). Previous work in yeast suggested that fusing a longer portion of ER

(251-596) to FLPe recombinase can increase activity, although this was not tested in

mammalian cells30. We synthesized this version (henceforth called FLPoER251) and

verified that it was more responsive to 4-OHT treatment and that its activity was not

increased by estrogen (E2) treatment (Extended Data Figure 5a). FLPoER251 was

leakier than FLPoER, but because it was one of three elements in the RM, we chose

to trade-off increased leakiness for increased responsiveness to 4-OHT and

proceeded with FLPoER251.

Creating spliced FLPoER251 for the RM: Next, we assembled a Rosa26 targeting

vector, which consisted of pTRE:Lox-STOP-Lox (LSL):FLPoER251 (Extended Data

Figure 5b), 2x CGG insulator, and the NM. Upon assembly, however, we discovered

that the NM in this construct was being recombined by FLPo activity in E coli

(Extended Data Figure 5c; note neither the LSL nor the ER251 prevented FLPo activity

in bacteria). Thus, we needed to redesign the RM so that FLPo was not expressed in

bacteria. We considered introducing an intron into the sequence of FLPoER251 (51), as

this would have prevented FLPo activity in bacteria. However, we became concerned

that low-level expression of FLPoER251 (through the LSL site) could lead to leaky

activation of the neoantigen in future applications. Therefore, we designed an

approach where FLPoER251 also required recombination before it could produce a

functional protein (Figure 1a).

To generate the inversion-inducible FLPoER251 construct, we first split FLPo into

N- and C-terminal halves. Based on its crystal structure, we decided to split FLPo

between a-helices I and J, which would split the catalytic residues across two exons.

We introduced splicing sites as before, taking advantage of K285 and D286 and

engineering an intron with SA and SD sequences from intron 2 of mouse p53. Here,

exon 2 was inverted relative to exon 1 and placed between non-compatible lox sites

(responsive to Cre-recombinase;52). Downstream of exon 2, we included a poly A

sequence from herpes simplex virus (HSV), which is predicted to terminate

transcription in both the 5’ -> 3’ and 3’ -> 5’ directions. Thus, in the OFF state, the HSV

poly A was oriented in such a manner that it should terminate the RNA transcript for

N-terminal FLPoER251, while in the ON state it would terminate the RNA transcript of

the full-length FLPoER251.

For integration into the larger construct, we reoriented the RM such that it would

run 3’ to 5’ relative to the NM. This meant that the pTRE-tight promoter would be next

to the 2x CGG insulator and the Rosa promoter (outside of the construct) would be

transcribed in the opposite direction of the RM. To prevent issues with transcription in

both directions and to allow for selection of clones after integration, we inserted a

cassette for puromycin resistance in the 5’-most end of the RM (Extended Data

Figure 6). This consisted of the PGK promoter, the puromycin resistance gene and

the HSV poly A sequence. In the OFF configuration, this element would provide

puromycin resistance and also terminate the endogenous transcript from the Rosa 26

promoter. Moreover, after Cre-mediated recombination in the RM, this puro resistance

cassette would be genetically deleted.

Supplementary Information references (46-52)

46. Kerppola, T.K. Bimolecular fluorescence complementation (BiFC) analysis as

a probe of protein interactions in living cells. Annu Rev Biophys 37, 465-487 (2008).

47. Schrempf, S., Froeschke, M., Giroglou, T., von Laer, D. & Dobberstein, B.

Signal peptide requirements for lymphocytic choriomeningitis virus glycoprotein C

maturation and virus infectivity. J Virol 81, 12515-12524 (2007).

48. van der Most, R.G. et al. Identification of Db- and Kb-restricted subdominant

cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice.

Virology 240, 158-167 (1998).

49. Puglielli, M.T. et al. In vivo selection of a lymphocytic choriomeningitis virus

variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb. J Virol

75, 5099-5107 (2001).

50. Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase

activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res

Commun 237, 752-757 (1997).

51. Kaczmarczyk, S.J. & Green, J.E. A single vector containing modified cre

recombinase and LOX recombination sequences for inducible tissue-specific

amplification of gene expression. Nucleic Acids Res 29, E56-56 (2001).

52. Stern, P. et al. A system for Cre-regulated RNA interference in vivo. Proc Natl

Acad Sci U S A 105, 13895-13900 (2008).

NM sequence:

Pre-FLPo DNA sequence: ACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCTAGGTGCAAAATCCCCAACGACCTGAAACAGAAAGTTATGAACCACAAAGGTGAGTACTAAGTACCGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGACCGGGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTAGCATCGTTACGCGTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGGATCCGTACTCACCTTGTAAATGTCAGGGCCGTTCAAACCCTTGTCATCGTCGTCCTTGTAGTCGATCCCGCATGTTGCAAAGTTGTACACAGCTGGGGAAAAAAAAGGGACAGGATAAGTATGACATCATCAAGGAAACCCTGGACTACTGCGCCCTGGGCCCGAAGTTCCTATACTATTTGAAGAATAGGAACTTCGGAATAGGAACTTCAAGCTTGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATAAGCTTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGGCGCGCCGCAGGACAACTGCATGGGGGCTGGGATGGGCATAAGAATAAATGTCTATGTGGACAGCCTTAACCACTTCAGCCATGACCTCTATGTATGTTTCTAACCCCACAGGGAGTTTATCAGTTTAAATCCGTTGAGTTTGACGCAGCCGCAGACCTGTCTATCTGTCGAAGATCCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA Post FLPo DNA sequence: ACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCTAGGTGCAAAATCCCCAACGACCTGAAACAGAAAGTTATGAACCACAAAGGTGAGTACTAAGTACCGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGACCGGGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCGGGCCCAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCCAGCTGTGTACAACTTTGCAACATGCGGGATCGACTACAAGGACGACGATGACAAGGGTTTGAACGGCCCTGACATTTACAAGGTGAGTACGGATCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCGGCGCGCCGCAGGACAACTGCATGGGGGCTGGGATGGGCATAAGAATAAATGTCTATGTGGACAGCCTTAACCACTTCAGCCATGACCTCTATGTATGTTTCTAACCCCACAGGGAGTTTATCAGTTTAAATCCGTTGAGTTTGACGCAGCCGCAGACCTGTCTATCTGTCGAAGATCCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

Deconstructed: Part I- Exon 1: Codes amino acids 1-155: N-terminal GFP: Kozak sequence + Amino acids 1-155 of eGFP. Silent mutation of C465T ACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCT Part II- Exon 1: Codes amino acids 156-170: N-terminal linker from BiFC RCKIPNDLKQKVMNH AGGTGCAAAATCCCCAACGACCTGAAACAGAAAGTTATGAACCAC Part III- Exon 1 and intron 1: Codes for amino acid 171 and one nucleotide of amino acid 172 (causes frame shift): Splice donor sequence (underlined). Also includes in frame TAA to truncate any read-through translation if not spliced. Continues with intron 1 AAAGGTGAGTACTAAGTACCGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGACCGG Part IV- Inversion module contains non-coding sequence comprising intron 1 pre flpo and introns 1/exon 2/intron 2 after flpo. Described pre and post FLPo Pre-FLPo: non-coding sequence in 3 parts (I-III): GAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTAGCATCGTTACGCGTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGGATCCGTACTCACCTTGTAAATGTCAGGGCCGTTCAAACCCTTGTCATCGTCGTCCTTGTAGTCGATCCCGCATGTTGCAAAGTTGTACACAGCTGGGGAAAAAAAAGGGACAGGATAAGTATGACATCATCAAGGAAACCCTGGACTACTGCGCCCTGGGCCCGAAGTTCCTATACTATTTGAAGAATAGGAACTTCGGAATAGGAACTTCAAGCTTGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATAAGCTTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTC Intron 1 part I: contains Frt-F3>, multiple cloning site, and Frt-wt> sequences GGGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTAGCATCGTTACGCGTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGGATCCGT Intron 1 part II: contains non-coding(inverted) sequence for splice acceptor (red) GP34.66-43/FLAG/GP66-69 ACTCACCTTGTAAATGTCAGGGCCGTTCAAACCCTTGTCATCGTCGTCCTTGTAGTCGATCCCGCATGTTGCAAAGTTGTACACAGCTGGGGAAAAAAAAGGGACAGGATAAGTATGACATCATCAAGGAAACCCTGGACTACTGCGCCCTGGGCCC Intron 1 part III: contains <Frt-wt, SV40 poly A sequence (light blue), and <Frt-F3: GAAGTTCCTATACTATTTGAAGAATAGGAACTTCGGAATAGGAACTTCAAGCTTGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATAAGCTTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTC

Post FLPo: intron 1/ exon 2/ intron 2 in 3 parts (I-III): GAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCGGGCCCAGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCCCAGCTGTGTACAACTTTGCAACATGCGGGATCGACTACAAGGACGACGATGACAAGGGTTTGAACGGCCCTGACATTTACAAGGTGAGTACGGATCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTC Intron 1 part I: retains Frt-F3>. Sequences for multiple cloning site and Frt-wt> sequences are eliminated: GGGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCGGGCCC Intron 1 part II -> Exon 2: Splice acceptor from human adenovirus C: AGGGCGCAGTAGTCCAGGGTTTCCTTGATGATGTCATACTTATCCTGTCCCTTTTTTTTCC Extra “C” nucleotide to encode P if translated + GP34.66-43: (GP34.66-41 underlined). Amino acids 171.66-182 CCAGCTGTGTACAACTTTGCAACATGCGGGATC Sequence encoding FLAG: Amino acids 183-190 GACTACAAGGACGACGATGACAAG Sequence encoding GP61-69 + splice donor: Amino acids 191-199 GGTTTGAACGGCCCTGACATTTACAAGGTGAGT Intron 1 part III -> Intron 2: retains <Frt-wt. Sequences for <Frt-F3 and SV40 poly A are eliminated ACGGATCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTC Part V- contained in intron 1 pre flpo and intron 2 post flpo: intron/splice acceptor from mouse IgG2b gene (red):. GGCGCGCCGCAGGACAACTGCATGGGGGCTGGGATGGGCATAAGAATAAATGTCTATGTGGACAGCCTTAACCACTTCAGCCATGACCTCTATGTATGTTTCTAACCCCACAG Part VI- Exon 2 pre FLPo and exon 3 post FLPo sequences encoding GP70-80: GGAGTTTATCAGTTTAAATCCGTTGAGTTTGAC Splice pre FLPo, in-frame stop codon: ATG AAC CAC AAA GGG AGT TTA TCA GTT TAA Splice product post FLPo, sequence encodes GP61-80: Amino acids 191-210 GGT TTG AAC GGC CCT GAC ATT TAC AAG GGA GTT TAT CAG TTT AAA TCC GTT GAG TTT GAC Part VII- 3’ UTR pre FLPo and exon 3 post FLPo: sequences encoding C-terminal linker from BiFC. Amino acids 211-222. AAADLSICRRSA GCAGCCGCAGACCTGTCTATCTGTCGAAGATCCGCC

Part VII- 3’ UTR pre FLPo and exon 3 post FLPo: sequences encoding C-terminal portion of eGFP. Amino acids 156-239 of eGFP. Amino acids 222-306 of construct. GACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA Products: cDNA eGFP: 1-720 ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA Amino acids eGFP: 1-239 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK- cDNA NINJA pre-FLPo: 1-838 ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCTAGGTGCAAAATCCCCAACGACCTGAAACAGAAAGTTATGAACCACAAAGGGAGTTTATCAGTTTAAATCCGTTGAGTTTGACGCAGCCGCAGACCTGTCTATCTGTCGAAGATCCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA Amino acids NINJA Pre-FLPo: 1-176* MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMARCKIPNDLKQKVMNHKGSLSV- cDNA NINJA Post-FLPo: 1-921 ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCTAGGTGCAAAATCCCCAACGACCTGAAACAGAAAGTTATGAACCACAAAGCTGTGTACAACTTTGCAACATGCGGGATCGACTACAAGGACGACGATGACAAGGGTTTGAACGGCCCTGACATTTACAAGGGAGTTTATCAGTTTAAATCCGTTGAGTTTGACGCAGCCGCAGACCTGTCTATCTGTCGAAGATCCGCCGACAAGCAGAAG

AACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA Amino acids NINJA Post-FLPo: 1-305 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMARCKIPNDLKQKVMNHKAVYNFATCGIDYKDDDDKGLNGPDIYKGVYQFKSVEFDAAADLSICRRSADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK-

NINJA Targeting Construct structure and sequence:

Feature name Location

Size

(bp) Directionality Type

AmpR promoter 109..137 29 == promoter

AmpR 179..1039 861 => CDS

Required for secretion to the

periplasm 179..247 69 => sig_peptide

PCR LoxP F 205..225 21 <= primer_bind

EuMyc-2 478..500 23 <= primer_bind

pMB1 ori 1105..1803 699 => rep_origin

ori 1134..1816 683 == rep_origin

repeat region 1147..1184 38 == repeat_region

pUC origin 1184..1857 674 == conflict

ori 1224..1859 636 == misc_feature

Lac O 2156..2178 23 == misc_feature

M13 pUC rev Primer 2165..2187 23 <= primer_bind

M13 Reverse 2184..2204 21 <= primer_bind

Left Homology Arm 2278..3075 798 == misc_recomb

MCS 3093..6153 3061 == misc_feature

LacZ 3093..3137 45 <= misc_feature

LoxpOrange<-- 3143..3176 34 => misc_recomb

SV40 pA 3209..3261 53 == polyA_signal

SV40 pA 3262..3262 1 == polyA_signal

Puro resistance 3263..3865 603 <= misc_feature

pGK 3891..4272 382 <= promoter

LoxpBlue<-- 4407..4440 34 => misc_recomb

SA 4447..4497 51 => misc_feature

cFlpo 4498..4947 450 == misc_feature

ER251 no atg 4954..5988 1035 == misc_feature

SV40 pA 5989..6042 54 == polyA_signal

LoxpOrange--> 6071..6104 34 <= misc_recomb

Lox Blue 6119..6152 34 <= misc_feature

SD 6160..6171 12 <= misc_feature

n-Flpo 6169..7023 855 <= misc_feature

pTRE 7046..7374 329 <= promoter

TRE 7047..13'487 6441 == misc_feature

CGG insulator 7413..8608 1196 == misc_feature

CGG insulator 8623..12'138 3516 == misc_feature

CAG promoter 9862..11'562 1701 => promoter

CMV enhancer 9862..10'225 364 == enhancer

Chicken B-Actin intron 10'506..11'469 964 == misc_feature

Intron 2 10'709..11'469 761 == intron

Rabbit Globbin Intron 11'470..11'562 93 == misc_feature

Intron_2 11'470..11'514 45 == intron

N-sGFP 11'566..12'028 463 == misc_feature

N-Link 12'029..12'075 47 == misc_feature

SD 12'076..12'087 12 => misc_feature

intron 1 12'095..12'133 39 == intron

Frt-F3+ 12'138..12'185 48 => misc_feature

Frt-F3+ 12'139..12'186 48 => misc_feature

Frt 12'237..12'497 261 => misc_feature

SD 12'291..12'301 11 <= misc_feature

GP34/FLAG/GP60-69 12'299..12'378 80 <= misc_feature

SA 12'384..12'443 60 <= misc_feature

Frt-F3- 12'450..12'497 48 <= misc_feature

SV40 poly A 12'503..12'693 191 == polyA_signal

SV40 poly A 12'503..12'693 191 == polyA_signal

Frt-WT- 12'694..12'741 48 <= misc_feature

Int 2 12'750..12'854 105 == intron

69-80 12'855..12'887 33 == misc_feature

C-Link 12'888..12'920 33 == misc_feature

C-sGFP 12'920..13'178 259 == misc_feature

GFP Inf F Age 12'974..12'993 20 => primer_bind

SP6 promoter 13'204..13'223 20 <= promoter

SP6 promoter 13'204..13'223 20 => promoter

SP6 promoter 13'205..13'224 20 => promoter

SP6 promoter 13'205..13'224 20 <= promoter

SP6 RNA polymerase

transcription initiation site 13'205..13'205 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'205..13'205 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'206..13'206 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'206..13'206 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'207..13'207 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'207..13'207 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'208..13'208 1 == misc_feature

SP6 RNA polymerase

transcription initiation site 13'208..13'208 1 == misc_feature

BGH pA 13'226..13'457 232 == polyA_signal

BGH\reverse\priming\site 13'244..13'261 18 == misc_feature

BGH\reverse\priming\site 13'244..13'261 18 == misc_feature

BGH rev primer 13'244..13'261 18 <= primer_bind

pA 13'246..13'473 228 == polyA_signal

Right Homology Arm 13'502..14'313 812 == misc_recomb

CreER#2 13'523..13'545 23 <= primer_bind

LacZ#1 13'876..13'896 21 <= primer_bind

EuMyc-1 14'440..14'461 22 => primer_bind

LacZ alpha 14'444..14'512 69 == misc_feature

f1 Ori 14'531..14'837 307 => mat_peptide

AACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGC

ACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT

CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTG

AAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT

GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAA

GATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAA

CAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAG

CACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA

AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC

ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAG

TGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT

CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA

CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAG

CGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACT

GGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGC

GGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTA

TTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAG

TCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACT

GATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT

TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT

CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT

AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC

TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAG

CTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT

ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA

CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG

CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG

CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG

AACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCA

CGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG

AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATA

GTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGT

CAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTT

CCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGAT

TCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAG

CCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC

AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC

ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTG

AGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGT

ATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGAC

CATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGA

GCTCTGTTTTGGTTGGCGTAAGGCGCCTGTCAGTTAACGGCAGCCGGAGTGCG

CAGCCGCCGGCAGCCTCGCTCTGCCCACTGGGTGGGGCGGGAGGTAGGTGG

GGTGAGGCGAGCTGGACGTGCGGGCGCGGTCGGCCTCTGGCGGGGCGGGG

GAGGGGAGGGAGGGTCAGCGAAAGTAGCTCGCGCGCGAGCGGCCGCCCACC

CTCCCCTTCCTCTGGGGGAGTCGTTTTACCCGCCGCCGGCCGGGCCTCGTCG

TCTGATTGGCTCTCGGGGCCCAGAAAACTGGCCCTTGCCATTGGCTCGTGTTC

GTGCAAGTTGAGTCCATCCGCCGGCCAGCGGGGGCGGCGAGGAGGCGCTCC

CAGGTTCCGGCCCTCCCCTCGGCCCCGCGCCGCAGAGTCTGGCCGCGCGCC

CCTGCGCAACGTGGCAGGAAGCGCGCGCTGGGGGCGGGGACGGGCAGTAGG

GCTGAGCGGCTGCGGGGCGGGTGCAAGCACGTTTCCGACTTGAGTTGCCTCA

AGAGGGGCGTGCTGAGCCAGACCTCCATCGCGCACTCCGGGGAGTGGAGGGA

AGGAGCGAGGGCTCAGTTGGGCTGTTTTGGAGGCAGGAAGCACTTGCTCTCCC

AAAGTCGCTCTGAGTTGTTATCAGTAAGGGAGCTGCAGTGGAGTAGGCGGGGA

GAAGGCCGCACCCTTCTCCGGAGGGGGGAGGGGAGTGTTGCAATACCTTTCT

GGGAGTTCTCTGCTGCCTCCTGGCTTCTGAGGACCGCCCTGGGCCTGGGAGA

ATCCCTTCCCCCTCTTCCCTCGTGATCTGCAACTCCAGTCTTTCTCCCGGTGCT

AGCTACTCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCC

CGGGATAACTTCGTATAGTACACATTATACGAAGTTATGGCTAAGCTTTGTGCC

ATGCCATTGATGCTAGGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCT

TTTTATTGCCACGCGTTCAGGCACCGGGCTTGCGGGTCATGCACCAGGTGCGC

GGTCCTTCGGGCACCTCGACGTCGGCGGTGACGGTGAAGCCGAGCCGCTCGT

AGAAGGGGAGGTTGCGGGGCGCGGAGGTCTCCAGGAAGGCGGGCACCCCGG

CGCGCTCGGCCGCCTCCACTCCGGGGAGCACGACGGCGCTGCCCAGACCCTT

GCCCTGGTGGTCGGGCGAGACGCCGACGGTGGCCAGGAACCACGCGGGCTC

CTTGGGCCGGTGCGGCGCCAGGAGGCCTTCCATCTGTTGCTGCGCGGCCAGC

CGGGAACCGCTCAACTCGGCCATGCGCGGGCCGATCTCGGCGAACACCGCCC

CCGCTTCGACGCTCTCCGGCGTGGTCCAGACCGCCACCGCGGCGCCGTCGTC

CGCGACCCACACCTTGCCGATGTCGAGCCCGACGCGCGTGAGGAAGAGTTCT

TGCAGCTCGGTGACCCGCTCGATGTGGCGGTCCGGATCGACGGTGTGGCGCG

TGGCGGGGTAGTCGGCGAACGCGGCGGCGAGGGTGCGTACGGCCCTGGGGA

CGTCGTCGCGGGTGGCGAGGCGCACCGTGGGCTTGTACTCGGTCATGGTNAA

GCTTGGGCTGCAGGTCGAAAGGCCCGGAGATGAGGAAGAGGAGAACAGCGCG

GCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGGGCCTCCGGAGGACCTTC

GGGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCCCCCGGACCCAC

CCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGCAAAGCTGCTATTGGCCG

CTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGCTGTCCATCTGCAC

GAGACTAGTGAGACGTGCTACTTCCATTTGTCACGTCCTGCACGACGCGAGCT

GCGGGGCGGGGGGGAACTTCCTGACTAGGGGAGGAGTAGAAGGTGGCGCGA

AGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTGGATGTGGAATG

TGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCT

AAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTACCT

AGCACAGCCTGAATGGATAACTTCGTATAGGATACCTTATACGAAGTTATACCG

GTTTCAAAAAATGGAAGGAAATCAGGAACTAACTCTCTGCTCTTGTTTTCCAGGA

CAACCTGGTGCGCAGCTACAACAAGGCCCTGAAGAAGAACGCCCCCTACCCCA

TCTTCGCTATCAAGAACGGCCCTAAGAGCCACATCGGCAGGCACCTGATGACC

AGCTTTCTGAGCATGAAGGGCCTGACCGAGCTGACAAACGTGGTGGGCAACTG

GAGCGACAAGAGGGCCTCCGCCGTGGCCAGGACCACCTACACCCACCAGATC

ACCGCCATCCCCGACCACTACTTCGCCCTGGTGTCCAGGTACTACGCCTACGA

CCCCATCAGCAAGGAGATGATCGCCCTGAAGGACGAGACCAACCCCATCGAG

GAGTGGCAGCACATCGAGCAGCTGAAGGGCAGCGCCGAGGGCAGCATCAGAT

ACCCCGCCTGGAACGGCATCATCAGCCAGGAGGTGCTGGACTACCTGAGCAG

CTACATCAACAGGCGGATTTCCGTACGCGGATCCAAAGGTGGGATACGAAAAG

ACCGAAGAGGAGGGAGAATGTTGAAACACAAGCGCCAGAGAGATGATGGGGA

GGGCAGGGGTGAAGTGGGGTCTGCTGGAGACATGAGAGCTGCCAACCTTTGG

CCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCT

GACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCT

ATTCCGAGTATGATCCTACCAGACCCTTCAGTGAAGCTTCGATGATGGGCTTAC

TGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGG

GTGCCAGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTTGAATGT

GCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTGGCGCTCCATGGAGCACCC

AGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATG

TGTAGAGGGCATGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGT

TCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTCAAATCTATTATTT

TGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAG

AGAAGGACCATATCCACCGAGTCCTGGACAAGATCACAGACACTTTGATCCACC

TGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCTGGCCCA

GCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGC

ATCTGTACAGCATGAAGTGCAAGAACGTGGTGCCCCTCTATGACCTGCTGCTG

GAGGCGGCGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCAT

CCGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCG

CATTCCTTGCAAAAGTATTACATCACGGGGGAGGCAGAGGGTTTCCCTGCCAC

AGCTTGAACGCGTGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTC

GTTTGTTCGGCGCGCCAGTAACATGTAGTAGTAAACATAACTTCGTATAATGTG

TACTATACGAAGTTATGGCTTCTAGCTGAGATAACTTCGTATAAGGTATCCTATA

CGAAGTTATGGATCTATTAACTCACCTTCAGCAGCTGGTACTCCTGCTTGTTGC

TGCTGCTGTTGCCGGTCCTGTTCACTCTCTTCAGCACGGGCTCGCTGTTCCTCA

GGAACTCGTCCAGGTACACCAGGGGGTCGATCCTGCCTCTGGCGCTGAAAAAG

TAGATGTGCCTGGACACGCTTGTCTTGGTCTCGGTCACCAGGCACTGAATGAT

CACGCCCAGGTACTTGTTCTGCACCAGCTTGAAGCTCTTGGGGTCCACGTTCTT

GATGTCGCTGAACCTGCCGCAGTTGATGAATGTGGCCAGGAACAGGAACTGGT

ACAGGGTCTTGGTCTTGGTGAACCTGCTGGTGTACTCGAAGCTGTTCAGGATCT

TCTCGGTGATCTCCCAGATGCTCTCGCCCTCGGACAGCAGGGCCTTCAGCATC

TTCTTGCTGTGGCTGTTGCCCTTGTCGGCCTCCTCGCTGCTCTCGAACTGCAG

CTGCAGGCTGGACACGATGTCGGTGATGTCGCTCTGGTGCTTCTGGCCGTTGT

AAGGGATGATGGTGAACTCCCAGGCGGGGATCAGCTTCTTCAGGCTGGCCTCC

AGGATGGTGGCCTTCTGGGTCTTGTACTTGAACTGCAGGCTCTTGTTCACGATG

TCGAAGCTCAGGCTGTTGCTGATGATGGTGTTGTAGCTCATGAAGGTGGCCCT

CTTGATGGCGGTGCCGTTGTGGGTGATCATCCAGCACAGGTAGGTCAGCTCGG

CGGCACAGCTGGCGATCTTCTCGCCGCTGGGCCTCTCGAATCTCTCCACGAAC

TGCCGCACCAGCACCTTGGGGGGGGTCTTGCACAGGATGTCGAACTGGCTCAT

CACCTTCCTCTTCTTCTTAGGAGCCATGGTGTCGAGTGACTTACGTATTCTCCA

GGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGGCCTCCCACCGTAC

ACGCCTACCTCGACATACGTTCTCTATCACTGATAGGGAGTAAACTCGACATAC

GTTCTCTATCACTGATAGGGATAAACTCGACATACGTTCTCTATCACTGATAGG

GAGTAAACTCGACATACGTTCTCTATCACTGATAGGGAGTAAACTCGACATACG

TTCTCTATCACTGATAGGGAGTAAACTCGACATCGTTCTCTATCACTGATAGGG

AGTAAACTCGACATACGTTCTCTATCACTGATAGGGAGTAAACTCGACATATCG

AAACCGTCGCGGCCGCCAGTGTGATGGATGACTCTAGAGGGACAGCCCCCCC

CCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGC

GAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGC

CGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCT

TTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATA

CGGGGAAAAAGCTTTAGGCTGAAAGAGAGATTTAGAATGACAGAATCATAGAAC

GGCCTGGGTTGCAAAGGAGCACAGTGCTCATCCAGATCCAACCCCCTGCTATG

TGCAGGGTCATCAACCAGCAGCCCAGGCTGCCCAGAGCCACATCCAGCCTGG

CCTTGAATGCCTGCAGGGATGGGGCATCCACAGCCTCCTTGGGCAACCTGTTC

AGTGCGTCACCACCCTCTGGGGGAAAAACTGCCTCCTCATATCCAACCCAAAC

CTCCCCTGTCTCAGTGTAAAGCCATTCCCCCTTGTCCTATCAAGGGGGAGTTTG

CTGTGACATTGTTGGTCTGGGGTGACACATGTTTGCCAATTCAGTGCATCACGG

AGAGGCAGATCTTGGGGATAAGGAAGTGCAGGACAGCATGGACGTGGGACAT

GCAGGTGTTGAGGGCTCTGGGACACTCTCCAAGTCACAGCGTTCAGAACAGCC

TTAAGGATAAGAAGATAGGATAGAAGGACAAAGAGCAAGTTAAAACCCAGCATG

GAGAGGAGCACAAAAAGGCCACAGACACTGCTGGTCCCTGTGTCTGAGCCTGC

ATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAG

AGATACAGCTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTA

GATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCC

AAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCA

CATCCCCACAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTG

GGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAAT

CCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTC

CAGGACGGAGTCAGTGAGGATGGGGCTCTAGAGGGACAGCCCCCCCCCAAAG

CCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCG

CCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAG

CGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCT

GAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAA

AAAGCTTTAGGCTGAAAGAGAGATTTAGAATGACAGAATCATAGAACGGCCTGG

GTTGCAAAGGAGCACAGTGCTCATCCAGATCCAACCCCCTGCTATGTGCAGGG

TCATCAACCAGCAGCCCAGGCTGCCCAGAGCCACATCCAGCCTGGCCTTGAAT

GCCTGCAGGGATGGGGCATCCACAGCCTCCTTGGGCAACCTGTTCAGTGCGTC

ACCACCCTCTGGGGGAAAAACTGCCTCCTCATATCCAACCCAAACCTCCCCTGT

CTCAGTGTAAAGCCATTCCCCCTTGTCCTATCAAGGGGGAGTTTGCTGTGACAT

TGTTGGTCTGGGGTGACACATGTTTGCCAATTCAGTGCATCACGGAGAGGCAG

ATCTTGGGGATAAGGAAGTGCAGGACAGCATGGACGTGGGACATGCAGGTGTT

GAGGGCTCTGGGACACTCTCCAAGTCACAGCGTTCAGAACAGCCTTAAGGATA

AGAAGATAGGATAGAAGGACAAAGAGCAAGTTAAAACCCAGCATGGAGAGGAG

CACAAAAAGGCCACAGACACTGCTGGTCCCTGTGTCTGAGCCTGCATGTTTGAT

GGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAG

CTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCA

TACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAA

CCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCA

CAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTG

TGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGA

CCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGA

GTCAGTGAGGATGGGGCTCTAGAGGATCTAGCCGCGTTGACATTGATTATTGA

CTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGA

GTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG

ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG

GGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGG

CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACG

GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA

CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGC

CCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTG

TATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGG

GCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGG

CGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT

TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC

GGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTC

GCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGG

CGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCT

CGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTT

TGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGG

AGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCG

GCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGG

CGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGG

GGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTG

TAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCG

GGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGG

GGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCG

GGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTC

GAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGC

GCAGGGACTTCCTTTGTCCCAAATCTGGCGGAGCCGAAATCTGGGAGGCGCC

GCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCCGGCAGGAA

GGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTC

CATCTCCAGCCTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGAC

GGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTC

TGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCT

GGTTATTGTGCTGTCTCATCATTTTGGCAAAACCATGGTGAGCAAGGGCGAGGA

GCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC

GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCA

AGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCC

ACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA

CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC

AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAG

GTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCG

ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC

AGCCACAACGTCTATATCATGGCTAGGTGCAAAATCCCCAACGACCTGAAACAG

AAAGTTATGAACCACAAAGGTGAGTACTAAGTACCGGAGTCGCTGCGTTGCCTT

CGCCCCGTGCCCCGCTCCGACCGGGAAGTTCCTATTCCGAAGTTCCTATTCTT

CAAATAGTATAGGAACTTCGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCT

AGCATCGTTACGCGTGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATA

GGAACTTCGGATCCGTACTCACCTTGTAAATGTCAGGGCCGTTCAAACCCTTGT

CATCGTCGTCCTTGTAGTCGATCCCGCATGTTGCAAAGTTGTACACAGCTGGG

GAAAAAAAAGGGACAGGATAAGTATGACATCATCAAGGAAACCCTGGACTACTG

CGCCCTGGGCCCGAAGTTCCTATACTATTTGAAGAATAGGAACTTCGGAATAGG

AACTTCAAGCTTGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATT

GCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCA

TTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGT

AAAACCTCTACAAATGTGGTATGGCTGATTATAAGCTTGAAGTTCCTATACTTTC

TAGAGAATAGGAACTTCGGAATAGGAACTTCGGCGCGCCGCAGGACAACTGCA

TGGGGGCTGGGATGGGCATAAGAATAAATGTCTATGTGGACAGCCTTAACCAC

TTCAGCCATGACCTCTATGTATGTTTCTAACCCCACAGGGAGTTTATCAGTTTAA

ATCCGTTGAGTTTGACGCAGCCGCAGACCTGTCTATCTGTCGAAGATCCGCCG

ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAG

GACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCG

ACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT

GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGA

CCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGTCGAGCAT

GCATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATC

AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGT

GCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA

GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGG

TGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG

GGATGCGGTGGGCTCTATGGCTTTATACTACACCTAGTCCGCGGAGATGGGCG

GGAGTCTTCTGGGCAGGCTTAAAGGCTAACCTGGTGTGTGGGCGTTGTCCTGC

AGGGGAATTGAACAGGTGTAAAATTGGAGGGACAAGACTTCCCACAGATTTTCG

GTTTTGTCGGGAAGTTTTTTAATAGGGGCAAATAAGGAAAATGGGAGGATAGGT

AGTCATCTGGGGTTTTATGCAGCAAAACTACAGGTTATTATTGCTTGTGATCCG

CCTCGGAGTATTTTCCATCGAGGTAGATTAAAGACATGCTCACCCGAGTTTTAT

ACTCTCCTGCTTGAGATCCTTACTACAGTATGAAATTACAGTGTCGCGAGTTAG

ACTATGTAAGCAGAATTTTAATCATTTTTAAAGAGCCCAGTACTTCATATCCATTT

CTCCCGCTCCTTCTGCAGCCTTATCAAAAGGTATTTTAGAACACTCATTTTAGCC

CCATTTTCATTTATTATACTGGCTTATCCAACCCCTAGACAGAGCATTGGCATTT

TCCCTTTCCTGATCTTAGAAGTCTGATGACTCATGAAACCAGACAGATTAGTTAC

ATACACCACAAATCGAGGCTGTAGCTGGGGCCTCAACACTGCAGTTCTTTTATA

ACTCCTTAGTACACTTTTTGTTGATCCTTTGCCTTGATCCTTAATTTTCAGTGTCT

ATCACCTCTCCCGTCAGGTGGTGTTCCACATTTGGGCCTATTCTCAGTCCAGGG

AGTTTTACAACAATAGATGTATTGAGAATCCAACCTAAAGCTTAACTTTCCACTC

CCATGAATGCCTCTCTCCTTTTTCTCCATTTATAAACTGAGCTGGTACCCAATTC

GCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTC

GTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCC

CCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA

ACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTA

AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCG

CCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG

GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTG

CTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTG

GGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCT

TTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTA

TTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG

CTGATTT

Supplementary Table 1: Binding strength of potential neoantigens

Supplementary Table 2: PCR primer sequences

Table of PCR primersName Sequence (5’->3’) NotesGFP F AgeI 72 TACTACCGGTTACTACCATGGTGAGCAAGGGCGAGGAG

GFP int F Age 66 TACTACCGGTCGAGGACGGCAGCGTGCAGC

GP80 R XhoI 64 tagtCTCGAGTTAGTCAAACTCAACGGATTTAAAC

Rosa26 WT F CCCTCTTCCCTCGTGATCTG Genotype NINJA/NINJA-C

Rosa26 WT R ACATAGTCTAACTCGCGACACTG Genotype NINJA

Rosa26 Ninja R GTCGAGGTGCCCGAAGGAC Genotype NINJA

Rosa26 Ninja-C R GAATCTGCAGGGAGAGGAGTT Genotype NINJA-C

Rosa26 Ninja-F F AAATCCCCAACGACCTGAAAC Genotype NINJA-F

Rosa26 Ninja-F R CTGAAGTGGTTAAGGCTGTCCA Genotype NINJA-F

Rosa26 southern probe F GCAAAGGCGCCCGATAGAATAA

Rosa26 southern probe R CCGGGGGAAAGAAGGGTCAC

Supplementary Table 3: Key resources

REAGENTorRESOURCE SOURCE IDENTIFIERAntibodies

Rabbitanti-GFPpolyclonal(wholeGFPprotein) Invitrogen A-11122

Mouseanti-GFPmonoclonal(clone3E6,requirescorrect

foldingofGFP)

Invitrogen A-11120

Rabbitanti-GFPmonoclonal(cloneD5.1,N-terminusspecific) CellSignalingTechnology 2956

Mouseanti-mouse/ratThy1.1monoclonal(BV421) BioLegend 202529

Ratanti-mouseThy1.2monoclonal(PerCP) BioLegend 105322

Ratanti-mouseCD8monoclonal(FITC) BioLegend 100706

Ratanti-mouseCD4monoclonal(BV605) BioLegend 100451

Ratanti-mouseCD44monoclonal(BV605) BioLegend 103047

Rabbitanti-mouseCD3polyclonal Abcam ab5690

Ratanti-mouseIFNgmonoclonal(PE) BDBiosciences 554412

Ratanti-mouseTNFamonoclonal(PerCP-eF710) eBioscience 46-7321-80

Ratanti-mouseCD8monoclonal(BV605) BioLegend 100744

Ratanti-mouseCD44monoclonal(PE) BioLegend 103008

Ratanti-mouseVa2TCRmonoclonal(APC) BDBiosciences 560622

Armenianhamsteranti-mouseCD11cmonoclonal(BV605) Biolegend 117333

Mouseanti-mouseH-2Kbmonoclonal(APC) eBioscience AF6-88.5.5.3

Rabbitanti-Grp94polyclonal CellSignalingTechnology 2104

BacterialandVirusStrains

LCMV-Armstrong Expandedinhouse

Ad5CMVFLPo(hightiter) IowaVectorCore VVC-UofIowa-

530HT

Ad5CMVCre(hightiter) IowaVectorCore VVC-UofIowa-5-

HT

Ad5CMVeGFP IowaVectorCore VVC-UofIowa-5-

HT

BiologicalSamples

None

Chemicals,Peptides,andRecombinantProteins

XenoLightD-Luciferin-K+SaltBioluminescentSubstrate PerkinElmer 122799

L(+)-lysinemonohydrochloride FisherScientific AC125220010

PierceSodiummeta-Periodate ThermoFisherScientific 20504

Formaldehydesolution37wt.%inH2O Millipore-Sigma 252549

16%paraformaldehydeaqueoussolution ElectronMicroscopySciences 15710

4-hydroxytamoxifenpowder Millipore-Sigma T176

Doxycyclinehyclaterodentdiet EnvigoTeklad TD.120769

LCMVGP33-41peptide Anaspec AS-61296

DMSO americanBIO AB03091

CellTraceVioletProliferationkit Invitrogen C34557 CrystalViolet Millipore-Sigma C0775

DoxycyclineHyclate Millipore-Sigma D9891

4-hydroxytamoxifenready-madesolution Millipore-Sigma SML1666

RecombinanthumanIL-2 PeproTech 200-02-250ug

RIPAlysisbuffer Millipore-Sigma

HEPESbuffersolution(1M) ThermoFisherScientific 15630-080

CollagenaseIV WorthingtonBiochemical LS004189

DNaseI Millipore-Sigma 10104159001

1xRBCLysisBuffer eBioscience 00-4333-57

Tissue-TekOptimumCuttingTemperature(O.C.T.)compound VWR 25608-930

CriticalCommercialAssays

BDCytofix/Cytoperm BDBiosciences 554714

Lipofectamine3000TransfectionReagent Invitrogen L3000008 EasySepCD8Tcellnegativeisolationkit StemCell 18000

BCAAssaykit

DepositedData

None

ExperimentalModels:CellLines

RMA-Scells Dr.PeterCresswell,Yale CVCL_2180

293Tcells ATCC CRL-3216

DC2.4 Millipore-Sigma SCC14M

KP-C4A3D6NINJAcells Generatedinhouse

ExperimentalModels:Organisms/Strains

NINJAmice Thispaper

NINJA-Cmice Thispaper

NINJA-Fmice Thispaper

Oligonucleotides

Seesupplementaryinformationfiles

RecombinantDNA

NINJAversion1vector(pcDNA3) Thispaper

NINJAversion2vector(pcDNA3) Thispaper

NINJAversion3vector(pcDNA3) Thispaper

NINJAversion4vector(pcDNA3) Thispaper

NINJAversion5vector(pcDNA3) Thispaper

NINJAversion6vector(pcDNA3) Thispaper

NINJAversion7vector(pBluescript) Thispaper

NINJAversion7-Avector(pBluescript) Thispaper

NINJAtargetingvector(pzDonor) Thispaper

SoftwareandAlgorithms

FlowJoV10.0.8 FlowJo

Photoshop Adobe

PrismV8.3.0 GraphPadSoftware

Tetramers

H-2D(b)KAVYNFATMAPC(LCMVGP33-41) NIHTetramerCore

H-2D(b)FQPQNGQFIAPC(LCMVNP396-404) NIHTetramerCore

I-A(b)DIYKGVYQFKSVAPC(LCMVGP66-77) NIHTetramerCore