inducible de novo expression of neoantigens in tumor cells ...10.1038... · 2koch institute for...
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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: [email protected]; [email protected]
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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