natural human genetic variation determines basal and inducible … · 2019-10-23 · * ^ rosi...
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1
Supplementary Information for Natural Human Genetic Variation Determines Basal and Inducible Expression of PM20D1, an Obesity-Associated Putative Thermogenic Gene Kiara K. Benson, Wenxiang Hu, Angela H. Weller, Alexis H. Bennett, Eric R. Chen, Sumeet A. Khetarpal, Satoshi Yoshino, William P. Bone, Lin Wang, Joshua D. Rabinowitz, Benjamin F. Voight, Raymond E. Soccio Corresponding author: Raymond E. Soccio Email: [email protected] This PDF file includes:
Figures S1 to S9 Tables S1 and S2
www.pnas.org/cgi/doi/10.1073/pnas.1913199116
exon 6/7 exon 120.0
0.5
1.0
1.5
Mouse Pm20d1
Rel
ativ
e G
ene
Expr
essi
on (Q
-PC
R) Control
Rosi* ^
Pm20d1
Ucp1
(Slc41a1) (Rab29)
(Elmod2) (Mgat4d)
100kbPPARγ ChIP-seq:
Mouse inguinal white fat
Mouse 3T3-L1 adipocytes
A
B
Figure S1. Mouse Pm20d1 is not an adipose PPARγ target gene.(A) Wild type mice were treated with rosiglitazone versus control diet (n=5 per group), and geneexpression measured by qPCR in inguinal white adipose tissue. Pm20d1 expression was measuredwith two primer pairs located in different exons. Mean and SEM, *P<0.01, ^P<0.05 by 2-tailed type 2Students T-test. (B) PPARγ ChIP-seq was performed in mouse inguinal white adipose tissue or 3T3-L1 adipocytes, as reported in Soccio et al, 2017. Browser tracks show lack of PPARγ occupancynear Pm20d1 (top panel), in contrast to strong binding sites near Ucp1 (bottom panel).
A
B
0.0
0.5
1.0
1.5
Rel
ativ
e G
ene
Expr
essi
on
*
NS NS
Pre-AdDeveloping AdAd (no rosi)Ad (constant rosi)Ad + vehicleAd + rosi
ACER3
PM20D1 KRT79 PPARG2 PM20D1 KRT79 PPARG20
5
10
15
20
25
30
Nor
mal
ized
Gen
e Ex
pres
sion vehicle control
1µM rosiglitazone3µM pioglitazone
Subcutaneous WAT Omental WAT
*
*
*
*
* **
*
Primary Human Adipocytes
Figure S2. PM20D1 is activated by multiple PPARγ agonists in human adipocytes.(A) SGBS cells were cultured as in Figure 2A-C, and expression of ACER3 was assayed by qPCR.(B) Human fat biopsy samples were obtained from abdominal subcutaneous and omental depotsfrom the same individual, and primary adipocytes were differentiated from adipose stem cells.Mature adipocytes were treated with dmso (vehicle control) versus the two indicated TZD drugs, andexpression of PM20D1, KRT79, and PPARG2 was assayed by Q-PCR. N=3 wells per condition,mean and SEM, * P<0.01 vs control by T-test.
A
Bsubject int up int up int up int up int up int up int up
1 AG GG AA CC CT TT TT CC GG AA CC CC CC TT2 AG GG AA AA TT TT TT CC GG AA CC CT CC TT4 AG GG AA AC TT TT TT CC GG AA CC CC CC TT5 GG GG AA AA TT TT TT CC GG AA CC CC CC TT7 AA GG AA AA TT TT TT CC GG AA CC CC CC TT8 AG GG AA CC TT TT TT CC GG AA CC CC CC TT10 AA GG AA AA TT TT TT CC GG AA CC CC CC TT12 AG GG AA AA TT TT TT CC GG AA CC CT CC TT13 GG GG AA AA TT TT TT CC GG AA CC CC CC TT14 AA GG AA AA TT TT TT CC GG AA CC CC CC TT15 AA GG AA AA TT TT TT CC GG AA CC CC CC TT16 AA GG AA AC CT TT TT CC GG AA CC CC CC TT17 AG GG AA AA TT TT TT CC GG AA CC CC CC TT
upSNP-332 Diff-336intSNP-120 *upSNP-180 intSNP-215 *Diff-229 Diff-242C
Figure S3. The segmental duplication in the human PM20D1 locus.(A) UCSC genome browser track showing the segmental duplication. (B) Description of this 1777bpduplication with >97% identity between the paralogous regions. (C) Genomic DNA from 13individuals was amplified using primers selective at position 336 for the intronic (int) or upstream(up) paralogous region, and PCR products were sequenced. Two other positions (229 and 242)differed between the paralogues, while four positions had true SNP with heterozygous individuals(yellow) identified at positions in the intronic (120 and 215) or upstream (180 and 332) paralogue.
int:TGTAGGGCAAAATTTGup:TGTAGGGCAAAGTTTGmus:TTTAGGGAAAATGTAG
int:TGTAGGGCAAAGATTGup:TGTAGGGCAAAGATTGmus:AGTGGAGTATAAAGTG
PPRE #2Consensus:
PPRE #1
Figure S4. Sequences of potential PPARγ binding sites near PM20D1.(A) Sequences from human PM20D1 gene body (intronic) and upstream region, as well as mousePm20d1 gene body (hg38 and mm10 genome assemblies) were aligned using CLUSTAL O (1.2.4)at default settings. Indicated are the second intron of PM20D1 (blue), the start and end of theluciferase reporter constructs (red), the three differences that distinguish the intronic and upstreamparalogs (orange), the true polymorphism in either paralog (green), and the PPAR binding motifs(PPREs, purple). (B) The sequences of the two PPREs are compared the consensus motif logo withdifferences bolded.
A
B
A
B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 260
5
10
15
20
25
Subject Number
vehiclerosiglitazone
Rel
ativ
e G
ene
Expr
essi
on
0 5 10 15 200
5
10
15
20
FABP4 fold induction
PM20
D1
fold
indu
ctio
n A/AA/CC/C
C
A/A A/C C/C0
5
10
15
20
25
Genotype
PM20
D1
fold
indu
ctio
n P=0.067
Figure S5. Genetic variation in human adipocyte PM20D1 induction by TZD drug.(A) Human adipose stem cell-derived adipocytes were cultured from 26 individuals and treated withrosi, and induction of FABP4 was consistently >3-fold (dotted line) by qPCR. Mean and SEM. (B)Scatterplot of fold-induction of PM20D1 (Fig. 5C) versus FABP4 (panel A) in these 26 adipocytesamples, with points colored by genotype at the PPRE SNP rs6667995. Adipocytes with the C/C(blue) or A/C (yellow) genotype generally have low induction of PM20D1 relative to FABP4. (C) Boxand whisker plot of fold rosi-activation of PM20D1 in all 26 adipocyte cultures, divided by rs6667995genotype. Median and range, with P-value calculated by 2-tailed type 2 Students T-test.
G/G G/A or A/A0
5
10
15
20
Genotype
up 3xnot up 3x
Rosi effecton PM20D1:
12/19(63%)
P>0.999
4/6(66%)N
umbe
r of I
ndiv
idua
ls
A/A A/C C/C0
2
4
6
8
10
12
14
16
18
20
Genotype atupstream PPRE SNP
PM20
D1
expr
essi
on
(n=30) (n=7) (n=3)
rs823075rs823074
rs708727
rs9438393rs17772159
rs823080
rs17772143
PM20D1SLC41A1
R-s
quar
ed v
alue
Com
bined Recom
bination Rate (cM
/Mb)
A/A A/G G/G0
1
2
3
4
5
Genotype at rs823080
AD
IPO
Qex
pres
sion
A B
C
Brain - CerebellumBrain - Cerebellar HemisphereSkin - Not Sun Exposed (Suprapubic)Skin - Sun Exposed (Lower leg)Cells - EBV-transformed lymphocytesBrain - Caudate (basal ganglia)TestisBrain - HypothalamusMuscle - SkeletalBrain - AmygdalaBrain - Anterior cingulate cortex (BA24)Brain - CortexCells - Transformed fibroblastsProstateLiverEsophagus - MucosaAdrenal GlandVaginaBrain - Nucleus accumbens (basal ganglia)Artery - TibialBrain - Frontal Cortex (BA9)Colon - SigmoidEsophagus - MuscularisWhole BloodUterusMinor Salivary GlandPituitaryColon - TransverseBrain - Putamen (basal ganglia)Brain - Substantia nigraSmall Intestine - Terminal IleumStomachLungPancreasEsophagus - Gastroesophageal JunctionBreast - Mammary TissueNerve - TibialArtery - CoronaryAdipose - SubcutaneousOvaryAdipose - Visceral (Omentum)Artery - AortaBrain - HippocampusBrain - Spinal cord (cervical c-1)SpleenThyroidHeart - Atrial AppendageHeart - Left Ventricle
Tissue
15412533541411714422510849188109136300132153358175106130388118203335369101851572461118012223738322021325136115238512231326711183146399264272
Samples
0.0270-0.00668-0.294-0.296-0.424-0.477-0.543-0.556-0.573-0.587-0.590-0.628-0.633-0.674-0.683-0.724-0.725-0.726-0.744-0.752-0.760-0.770-0.773-0.776-0.777-0.793-0.808-0.810-0.813-0.814-0.832-0.842-0.908-0.909-0.937-0.950-0.950-0.954-0.976-0.977-0.979-0.994-0.996-1.01-1.01-1.04-1.06-1.08
NES
0.816.0e-114.9e-101.3e-45.5e-55.6e-122.0e-52.6e-204.1e-59.0e-71.9e-71.8e-181.4e-95.9e-99.8e-278.3e-193.0e-92.0e-85.7e-322.0e-84.8e-175.4e-323.6e-451.0e-106.4e-92.5e-151.5e-302.4e-101.3e-71.8e-181.5e-421.1e-442.6e-419.9e-288.9e-405.0e-671.9e-212.3e-573.5e-229.4e-493.7e-438.9e-142.2e-146.9e-296.3e-703.9e-433.3e-56
p-value
0.000.000.000.000.8341.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.001.00
m-valueSingle-tissue eQTLNES (with 95% CI)
-1.0 -0.5 0.0
NES
Single-tissue eQTL p-valueversus Multi-tissue Posterior Probability
0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
m-value (Posterior Probability from METASOFT
-log1
0( S
ingl
e-tis
sue
eQTL
p-v
alue
)
DPPARγ site
Preads Ads Ads+Rosi0
20
40
60
80
100
SGBS cells
Met
hyla
tion
atPM
20D
1 TS
S (%
)E
F
rs1361754
Figure S6. Genetically variable PM20D1 expression in human tissues.(A) PM20D1 expression by qPCR in 40 subcutaneous white fat biopsies, divided by genotype at SNPrs6667995. (B) Linkage disequilibrium analysis showing six SNPs tightly linked to lead eQTL SNPrs708727, and weaker linkage to PM20D1 missense SNP rs1361754. A recombination hotspot (redrectangle) falls in the gene, such that the upstream PPARγ site SNP rs6667995 is not linked to these.(C) Expression of ADIPOQ was measured the same fat biopsies, divided by genotype at rs823080. (D)Screenshot of GTEx browser showing rs823080 significantly correlates with PM20D1 expression innearly all human tissues. (E) DNA methylation near PM20D1 start site was assayed by pyrosequencingin cultured SGBS cells, comparing preadipocytes (Preads), adipocytes (Ads), and rosi-treatedadipocytes (Ads+rosi). While PM20D1 expression changes drastically in these three cases (see Fig 2B),methylation is unchanged. (F) In cultured adipocytes, induction of PM20D1 by rosiglitazone did notcorrelate with genotype at eQTL SNP rs823080. Contingency P-value by two-sided Fisher’s exact test.
N-stea
royl Thr
N-stea
royl tau
rine
N-palmito
yl Val
N-palmito
yl Thr
N-palmito
yl tau
rine
N-palmito
yl Ser
N-palmito
yl Pro
N-palmito
yl Gly
N-oleoyl
taurin
e
N-oleoyl
Ser
N-oleoyl
Gly
N-oleoyl
Ala0
1
2
3
4
5
A/A G/GA
B
N-palmito
yl Gln
N-palmito
yl Gly
N-oleoyl
Gly
N-palmito
yl Ser
N-oleoyl
Ser
N-stea
royl Thr
N-palmito
yl Thr
N-oleoyl
taurin
e
N-palmito
yl tau
rine
N-stea
royl tau
rine
N-palmito
yl Val
0
1
2
3
4
5 SQ - A/ASQ - G/G
omental - A/Aomental - G/G * *
*
NS
Omental fat:
C
0 10 20 30 40 500
10
20
30
40
50
N-palmitoyl Ser
Omental
Subc
utae
nous
R2=0.797
0 10 20 30 400
10
20
30
40
N-oleoyl Gly
Omental
R2=0.603
0 5 10 15 200
5
10
15
20
N-oleoyl Ser
Omental
R2=0.802
D
Gln Gly
Leu/Ile Lys Pro Ser
Taurin
eThr
Val Gln Gly
Leu/Ile Lys Phe
Pro
Taurin
eThr
Val Gln Gly
Leu/Ile Lys Phe
Ser
Taurin
e0
1
2
3A/A A/G G/G
N-palmitoyl (16:0) AAs N-stearoyl (18:0) AAs N-oleoyl (18:1) AAs
Serum NAAs by rs823080 gentoype:
*
0 10 20 30 40 50 600
10
20
30
40
50
60
N-palmitoyl Thr
Omental
R2=0.491
Rel
ativ
e N
AA L
evel
Rel
ativ
e N
AA L
evel
Rel
ativ
e N
AA L
evel
Figure S7. N-acyl amino acid levels do not correlate with rs823080 genotype.(A) NAAs were assayed in 20 human omental fat samples, n=10 A/A (green) and n=10 G/G (orange).(B) NAAs were similarly assayed in 10 additional individuals, n=7 A/A (green) and n=3 G/G (orange),using paired omental (hatched bars) and subcutaneous (plain bars) fat samples from each. *P<0.01 bypaired 2-tailed type 2 Students T-test. (C) Scatterplots of subcutaneous versus omental NAA levels inthese 10 individuals, showing correlation by linear regression (dotted line) between the depots. (D)NAA levels were assayed in 21 human serum samples, n=6 A/A (green), n=7 A/G (yellow), and n=8G/G (orange). Mean and 95% confidence interval, *P<0.05 by 2-tailed type 2 Students T-test.
A
B
C
SNP alleles MAF major minorrs1772159 (G/A) 0.1358 G A G G A G A G A A Ars708727 (G/A) 0.1414 G A A G A A A G A A Grs823074 (T/C) 0.1412 T C C T C C C T C T Trs823075 (C/T) 0.1412 C T T C T T T C T C C
rs9438393 (A/G) 0.1408 A G G G A A G G A A Ars823080 (G/A) 0.1412 G A A A G G A A A G G
rs1772143 (T/A) 0.1380 T A A T T T T A A T TN= 4284 663 22 11 8 7 5 4 2 1 1
freq= 0.855 0.132 0.0122
rare haplotypesHaplotypes:
Type 2 DiabetesBody Mass Index HDL cholesterol
Figure S8. Human Genetics of the PM20D1 locus.(A) Haplotype analysis was performed on the seven linked expression SNPs for PM20D1 on 5,008human chromosomes using LDLink. (B) All catalogued GWAS SNPs were interrogated for pairwiselinkage to each other and the seven PM20D1 eSNPs (yellow, starred) using LDLink, and thedegree of linkage indicated by a heat map (white to red). GWAS SNPs and phenotypes with weaklinkage to the eSNPs are labelled on the left, while those with strong linkage are labelled on theright. (C) Locus zoom plots showing GWAS signals in this region, with SNPs colored by linkage tothe PM20D1 eSNP rs823080 (purple).
rs1772159rs708727 rs9438393rs823074
rs823075rs823080
rs1772143
rs1361754
0
0.603
G-T (high/Ile): 0.478G-C (high/Thr): 0.125A-C (low/Thr): 0.397A-T (low/Ile): 0
0.478
A/lowrs823080
G/high
R2=0.6022D’=1.0
0.397
rs13
6175
4
T/Ile
C/Thr 399 126
481
0.522
G/G(121)
G/A(141)
A/A(51)
T/T(65)
T/C(165)
C/C(83)
2
1
0
-1
-2Nor
mal
ized
PM
20D
1Ex
pres
sion 3
rs823080 rs1361754
A B
C
P=9.4x10-49 P=3x10-30
Figure S9. Linkage of the PM20D1 missense SNP rs1361754 to expression SNPs.(A) Pairwise linkage was calculated in European populations using LDLink for the missense SNPrs1361754 (T encoding Ile, C encoding Thr) and the lead expression SNP rs823080 (A with lowexpression, G with high). (B) Violin eQTL plots from GTEx showing association of genotypes ateach of these two SNPs with PM20D1 expression in human omental adipose tissue. Dottedhorizontal lines mark average expression in the rs823080-G/G genotype (similar to rs1361574-T/T)and the rs823080-A/A genotype (lower and less variable than rs1361574-C/C). (C) Locus zoom plotfrom GTEx showing SNPs associated with PM20D1 expression in human omental fat, with rs823080the most significant (green), other labelled highly linked SNPs similarly significant, but the missenseSNP rs1361754 (blue) less significant. The dotted box shows the weak association of rs823080 withexpression of other nearby genes further downstream of PM20D1 (RAB7L1, blue; NUCKS1, red).
Table S1. SNPs and Parolgous Differences in the PM20D1 segmental duplication
intron 2SNP or difference position intron upsteam intron upsteam intron upsteam note
difference 336 205,847,549 205,854,210 C C/T rs71524489 rs6668122 difference 1 in PCRSNP (upstream) 332 205,847,553 205,854,206 C C/T none rs71633566
difference 242 205,847,643 205,854,116 G A none rs61814918 difference 2difference 229 205,847,656 205,854,103 T C rs571503374 rs61814917 difference 3 in PPRE1
SNP (intron) 215 205,847,670 205,854,089 T/C T rs12756994 noneSNP (upstream) 180 205,847,705 205,854,054 A A/C rs549511193 rs6667995 SNP in PPRE2
SNP (intron) 120 205,847,765 205,853,994 G/A G rs112268616 rs372515785
chr1 position (hg38) base (ref/oth) dbSNP 150 annotation
Table S2. Primer Sequences.
Primer name Primer sequence Figure(s) Notehuman primers RPLP0_F GATGCCCAGGGAAGACAG 2B, 2C, S2A, S2B gene expression Q-RT-PCR, normalizer
RPLP0_R TCCCACAATGAAACATTTCGPM20D1_ex3_F ACCTGCTGATGGCTCACTTT 2B, S2B gene expression Q-RT-PCR
PM20D1_ex3_R GTTCTTGTCGTCCAGTGTGCPDK4_F AGGAGGTGGTGTTCCCCTGAGA 2C gene expression Q-RT-PCR
PDK4_R AACCAGCCAAAGGAGCATTCCGGACER3_F AACCTGATCATGATTATACCTCCAA S2A gene expression Q-RT-PCR
ACER3_R TGCTAAATAAGAAGCAATGTACCGKRT79_R CTCACCCGCACTATCCAGAG S2A gene expression Q-RT-PCR
KRT79_F ATCCAGATCCCCAAGCTTCTPPARG2_F AGCAAACCCCTATTCCATGCT S2A gene expression Q-RT-PCR
PPARG_R CACGGAGCTGATCCCAAAGTADIPOQ_F TCCACCTTCACAGGCTTTCT S6C gene expression Q-RT-PCR
ADIPOQ_R ATTGACTTTGGGGCTGTTTGPM20D1_up/int2_F CAAATCAGGGGCAAGGGGTG 3C, S3C non-selective forward PCR and sequencing primer
PM20D1_int2_336_R GTGTGTAAGGAGGTAGGGGTG reverse PCR primer, intron2-selective reverse at 336
PM20D1_up_336_R GTGTGTAAGGAGGTAGGGGTA reverse PCR primer, upstream-selective reverse at 336
ALB_ChIP_F TGGCACAATGAAGTGGGTAA 4B ChIP primers at negative control non-binding site
ALB_ChIP_R CTTACGTGCATCTCGACGAA PM20D1_ChIP_up/int_R TGATCAGAGGACAATCTAATTCC 4B ChIP non-selective reverse primer
PM20D1_ChIP_up_F GATGATTTAACATTTATTGAATGACACAT ChIP primer, upstream-selective at 242
PM20D1_ChIP_int_F GATGATTTAACATTTATTGAATGACACAC ChIP primer, intron2-selective at 242
PM20D1_up/in_pyroF GGAAATCCTAGTTCTACTTTTTGA 4C amplifies up/in non-selectively for pyrosequencing
PM20D1_up/in_pyroR_5bio [biotin]TCATCCAGCCATTGCATTAG amplifies up/in non-selectively for pyrosequencing
PM20D1_up/in_pyroS TTTAACATTTATTGAATGAC pyrosequencing to quantify nucleotide at 229
PM20D1_rep_F ctgagctcgctagcCAAATCAGGGGCAAGGGGTG 4D, 5B reporter Gibson cloining, non-selective forward primer
PM20D1_rep_up_R gccagatcttgatatccTGTAAGGAGGTAGGGGTAGG reporter Gibson cloining, reverse primer upstream-selective at 336
PM20D1_rep_int2_R gccagatcttgatatccTGTAAGGAGGTAGGGGTGGG reporter Gibson cloining, reverse primer intron2-selective at 336
PM20D1_mut_R TTTATACTCTAATGCAATGGC 4E wild type primer for reporter mutagenesis
PM20D1_mut_intAtoG_F CTCTCTCAAAcTTTGCCCTAC mutagenic primer, position 229 change A to G
PM20D1_mut_enhGtoA_F CTCTCTCAAAtTTTGCCCTAC mutagenic primer, position 229 change G to A
HPRT_F TGACACTGGCAAAACAATGCA 5C, S5A gene expression Q-RT-PCR, normalizer
HPRT_R GGTCCTTTTCACCAGCAAGCTPM20D1_ex1_F TAGTTTTCCCTACCGTCTCCAG 5C gene expression Q-RT-PCR
PM20D1_ex1_R GCGTTCCTCTTTGCTGAACTGFABP4_F TCATGAAAGGCGTCACTTCC S5A gene expression Q-RT-PCR
FABP4_R GCTTGCTAAATCAGGGAAAACAPM20D1_A/C_pyro_F_5bio [btn]TGACACATATGTAGGGCAAAGT 5D pyrosequencing to genotype DR1 SNP rs6667995
PM20D1_A/C_pyro_R CTTGGAAAAACTAAGCCACCATAPM20D1_A/C_pyro_S CACCATATGTAGGGCAArs823080_F TGCGCTAGTTCTCTCCCAGA 6A, 6B SNP genotyping assay
rs823080_R TGGTGATTGATGGGTGGTGGrs823080_SNaP CAGAAAAGAGGATTCCCTCPM20D1_CpGmeth_F GGTAGGGAAAATTAGGAGTAGTATAGT 6B Methylation bisulfutite pyrosequencing assay
PM20D1_CpGmeth_R [btn]CAAAACTACCCCRATAACCTAACAACAPM20D1_CpGmeth_S AAAATTAGGAGTAGTATAGTTATTA
mouse primers Actb_F CTCTGGCTCCTAGCACCATGAAGA 1A, 1C gene expression Q-RT-PCR, normalizer
Actb_R GTAAAACGCAGCTCAGTAACAGTCCGUcp1_ex5_F TCATCAACTCTCTGCCAGGAC 1A gene expression Q-RT-PCR
Ucp1_ex6_R CGCAGAAAAGAAGCCACAADio2_F CCAGACAACTAGCATGGCGT 1A gene expression Q-RT-PCR
Dio2_R GAAAATTGGCTGCCCCACACPm20d1_exon6_F CTCTTCAGCTCCCCCAAAGG 1A, S1A, 1C gene expression Q-RT-PCR
Pm20d1_exon7_R TCAATGGCCCTCCTCCAAACPm20d1_exon12_F GACACCCGACACTATGCCAA S1A gene expression Q-RT-PCR
Pm20d1_exon12_R CCACTGAAGTCCTGAGGGTTGyK_F CAAGCAGGACGATGCTTTTT 1C gene expression Q-RT-PCR
GyK_R CGTTGGGAAGAATTTCCATTPm20d1_int2_Fgib ctgagctcgctagccCTAAGGGCTAAGGGTGGCAG 4D reporter Gibson cloning
Pm20d1_int2_Rgib gccagatcttgatatccGCCTGCATGCCACATGTATG