Spreadsheet: 95d19c52-1b24-440a-9fcf-a9992e3081f5
Sheet: primers reporter constructs

Oligonucleotides used for PCR amplification and GeneArt Seamless cloning of the 3'UTR or exonic regions of candidate [NAME] miRNA targets into the pGL3 promoter vector. Columns show official gene symbol, amplified gene region (3'UTR/Exon), primer name including orientation (for = forward, rev = reverse), primer sequence (including a 16-17 bp overhang for Seamless cloning,marked in bold), and genomic coordinates of the primers ([NAME] for the human genome and nc_009333.1 for the KSHV genome)
Gene | 3'UTR/Exon | Primer Name | Sequence | genomic coordinates
ANXA2 | 3'UTR | ANXA2-pGL3-for | GCCGTGTAATTCTAGACAAGACACTAAGGGCGACTACCAGAAAGC | [NAME]; 60639888-60639860
ANXA2 | 3'UTR | ANXA2-pGL3-rev | GCTCGAAGCGGCCGGCCCATGTGTACCATTGTATCAGCGACCTGTCATT | [NAME]; 60638891-60638860
C1QBP | Exon | C1QBP_E2E6_pGL3_for | GCCGTGTAATTCTAGACCTAAGATGTCTGGAGGTTGGGAG | chr17 p13.2; 5341519-5341493
C1QBP | Exon | C1QBP_E2E6_pGL3_rev | GCTCGAAGCGGCCGGCCGCACATGTCTAGCTTCAGAGTAG | chr17 p13.2; 5336284-5336255
CEBPA | 3'UTR | CEBPA-pGL3-for | GCCGTGTAATTCTAGACTTGTGCCTTGGAAATGCAAACTCACC | chr19 q13.11; 33791963-33791937
CEBPA | 3'UTR | CEPBA-pGL3-rev | GCTCGAAGCGGCCGGCCAAGAAGAGAACCAAGCCGTCCTTC | chr19 q13.11; 33790935-33790912
HLA-C | 3'UTR | HLA-C_3UTR_pGL3_for | GCCGTGTAATTCTAGATGATTGGGACGTTTCGAGTGTGTG | chr6 p21.33; 31236405-31236432
HLA-C | 3'UTR | HLA-C_3UTR_pGL3_rev | GCTCGAAGCGGCCGGCCCTGACCACATAAAGCCCATGAAG | chr6 p21.33; 31236432-31236405
HLA-E | Exon | HLA-E_pGL3_for | GCCGTGTAATTCTAGAAGTTCTCAGGACTCAGAGGCTGGGAT | chr6 p21.33; 1750922-1750948
HLA-E | Exon | HLA-E_pGL3_rev | GCTCGAAGCGGCCGGCCAGCTCCAGAGACCACAGATCCAAGGAG | chr6 p21.33; 1753006-1753033
HMGA1 | 3'UTR | HMGA1-3'UTR-pGL3-for | GCCGTGTAATTCTAGAAAGAGGAGGGCATCTCGCAGGA | chr6 p21.31; 34212618-34212639
HMGA1 | 3'UTR | HMGA1-3'UTR-pGL3-rev | GCTCGAAGCGGCCGGCCAGGCTGGGACAAATCATGGCTACC | chr6 p21.31; 34213923-34213946
IRF2BP2 | 3'UTR | IRF2BP2_pGL3_for | GCCGTGTAATTCTAGAAGCTAGTGGAGAGGTCTATTGTCCCAGT | chr1 [NAME]; 234743021-234742994
IRF2BP2 | 3'UTR | IRF2BP2-pGL3_rev | GCTCGAAGCGGCCGGCCCACACATCCTTTAGTCACATTGCCATTAGTGA | chr1 [NAME]; 234739956-234739923
PTPN11 | Exon | PTPN11-pGL3-for | GCCGTGTAATTCTAGATATGGCGTCATGCGTGTTAGGAACGT | chr12 q24.13; 112919923-112919950
PTPN11 | Exon | PTPN11-pGL3-rev | GCTCGAAGCGGCCGGCCTGAGGAAATGAGTAATTGGGAAGCAGGTATTGTTGAG | chr12 q24.13; 112943780-112943816
STIP1 | Exon | STIP1-pGL3-for | GCCGTGTAATTCTAGAACACGAGGCAAATAACCCTCAACTGAAAGAG | [NAME]; 63961741-63961771
STIP1 | Exon | STIP1-pGL3-rev | GCTCGAAGCGGCCGGCCGTTCCTTTTGATGGCTTCTGTATAATGCTTCATG | [NAME]; 63967657-63967690
TP53INP1 | 3'UTR | TP53INP1-pGL3-for1 | GCCGTGTAATTCTAGACACAATGGCTGGGTTGTTCATCAGC | chr8 q22.1; 95942757-95942733
TP53INP1 | 3'UTR | TP53INP1-pGL3-rev1 | GCTCGAAGCGGCCGGCCACCTGTCACTCTTCACTCAGATGCC | chr8 q22.1; 95941700-95941676
TP53INP1 | 3'UTR | TP53INP1-pGL3-for2 | GCCGTGTAATTCTAGATGCTGTGTATCACATAATGCCTTGCC | chr8 q22.1; 95942547-95942522
TP53INP1 | 3'UTR | TP53INP1-pGL3-rev2 | GCTCGAAGCGGCCGGCCGGACAGAATTGGCGGGAAGGA | chr8 q22.1; 95941910-95941890
TPD52 | 3'UTR | [NAME] | GCCGTGTAATTCTAGATCAAACTTCTAGGTGACTTGAGAGTGGAACCT | chr8 q21.13; 80949731-80949700
TPD52 | 3'UTR | [NAME] | GCTCGAAGCGGCCGGCCACTCTAGCAATCAAGAATGGCAGCATG | chr8 q21.13; 80948928-80948902
[NAME] | 3'UTR | YWHAE-pGL3-for | GCCGTGTAATTCTAGACATTGTCACTGAGAACCACCAAATCTG | chr17 p13.3; 1248671-1248645
[NAME] | 3'UTR | YWHAE-pGL3-rev | GCTCGAAGCGGCCGGCCCTCAGTGTTCTTCACAACCAGACTACA | chr17 p13.3; 1247677-1247651
vIL6 | 3'UTR | vIL6-pGL3-for | GCCGTGTAATTCTAGACTAGTATTGTGTTCCCATACGC | HHV-8 17057-17078
vIL6 | 3'UTR | vIL6-pGL3-rev | GCTCGAAGCGGCCGGCCGATAAGTAAAGAGCGACGTGAC | HHV-8 17235-17214
vIL6 | 3'UTR | vIL6-pGL3-rev-short | GCTCGAAGCGGCCGGCCGATAAGTAAAGAG | HHV-8 17235-17227
Primers for introduction of seed match mutations using GeneArt Seamless cloning; mutated bases in the seed sequence (bold) are highlighted in red; overlapping sequence of the primers is highlighted in green; for PCR the forward mutagenesis primer was paired with the reverse primer originally used for GeneArt cloning of the fragment (above table), and the reverse mutagenesis primer was paired with the forward GeneArt cloning primer yielding two overlapping PCR products; for vIL6 the forward mutageneis primer was paired with a shortened version of the vIL6 reverse cloning primer (vIL6-pGL3-rev-short) to avoid sequence overlap between the two primers.
Gene | 3'UTR/exon | primer name | Sequence
ANXA2 mutant | 3'UTR | ANXA2mutF | gacgttacgataacccccaacctcattttag
ANXA2 mutant | 3'UTR | ANXA2mutR | GGTTATCGTAACGTCACCCTCACAGGGATG
YWHAE mutant | 3'UTR | YWHAEmutF | ttcacgataactgtgtcgtctccgtgccag
YWHAE mutant | 3'UTR | YWHAEmutR | CACAGTTATCGTGAAAAAGCCTCTATGTAGTCCTG
vIL6 mutant 1 | 3'UTR | vIL6mutF1 | gtatattaggcgtataagaaattttatgtcacgtcgct
vIL6 mutant 1 | 3'UTR | vIL6mutR1 | ATACGCCTAATATACTTTGCGTGTCTAGACAATAAAG
vIL6 mutant 2 | 3'UTR | vIL6mutF2 | gtatattaaaataataagaaattttatgtcacgtcgct
vIL6 mutant 2 | 3'UTR | vIL6mutR2 | [NAME]