Box: Oligo box D
1) NELF-A c-ter.3 2) NELF-A c-ter.4 3) NELF-A HDag.1 4) NELF-A HDag.2 5) Tailed-4 6) Tailed-5
 7)
8
9)
fcp-N1 R1
 10)
fcp-N3 Xho1
 11)
fcp-C1b R1
 12)
fcp-C2b Sal
 13) tat SF1 B.1 14)
tat SF1
B.2		 15)
tatSF1 N-A 5'		 16)
tatSF1 N-A 3'
17)
tatSF1 N-B 5'
 18)
tatSF1 N-B 3'
 19)
tatSF1 rrm 5'		 20)
tatSF1 rrm 3'		 21) tatSF1 N-A.2 5’ 22) tatSF1 N-A.2 3’ 23) Sfi1-A 24) Sfi1-B
25)
Gal4-fusion A.1		 26)
Gal4-fusion A.2		 27)
pGEX5' seq primer		 28)
pGEX3' seq primer		 29)
G-less +35/+10		 30)
G-less +344/+318
 31)
p70_
+35/+10		 32)
p70_+60/+40
33) FLAG-NELF-B.1 34) FLAG-NELF-B.2 35) FLAG-NELF-B.3 36) FLAG-NELF-B.4 37) NELF-A C-ter.1 38)
NELF-A C-ter.2		 39)
TR-3
 40)
41) RNAi-NELF-B.1 42) RNAi-NELF-B.2 43) RNAi-NELF-B.3 44) RNAi-NELF-B.4 45)
RNAi-NELF-B.5
 46)
NELF-A 3'UTR-1		 47)
NELF-A 3'UTR-2		 48)
NELF-A 3'UTR
49)
RNAi-NELF-D.1
 50)
RNAi-NELF-D.2
 51)
 RNAi-NELF-D.3
 52)
 RNAi-NELF-D.4
 		53)
 RNAi-NELF-A.1
 		54)
 RNAi-NELF-A.2
 55)
 RNAi-NELF-A.3
 		56)
 RNAi-NELF-A.4
57) Tailed-1 58) Tailed-2 59) Tailed-3 60) fcp pd1 61) fcp pd2 62) fcp brca1 63) fcp brca2 64)

Description of contents:

Row 1 (return to top)
1)
 NELF-A c-ter.3: gcgtagctagcCGTGGACTCTCTCTATCGAA
2)
 NELF-A c-ter.4: gcctaaagcttGCGTATGACCCTTGTGGA
3)
 NELF-A HDag.1: gcgtagctagcCTATGGGTTTCCATGCTG
4)
 NELF-A HDag.2: gcctaaagcttaTTCGGTGAATTCGATCA
5)
 Tailed-4: 5'- AAG CTT GGG CTG CAG GTC -3'.  Zhiqiang is using this to generate a tailed template of ~130 nucs.
6)
 Tailed-5: 5'- /5Phos/GAT CAA AAA AAA TTA -3'. Zhiqiang is using this to ad a tail that won't bind UpG near its end.
7)
8)
  
Row 2 (return to top)
9)
  fcp-N1 R1:ATG CGA ATT CCA TGC AGA AGA TAC CGG ACG A, for amplifying N-terminus of FCP1 and inserting it into R1/XhoI cut pGEX-4T3. (Tm=60.6)
10)
  fcp-N3 Xho1: GAT CCT CGA GCC GTA CAG CTG GAA GTG G,  for amplifying N-terminus of FCP1 and inserting it into R1/XhoI cut pGEX-4T3. (Tm=61.8)
11)
  fcp-C1b R1: GAT CGA ATT CAC CCA CGC AGA TGA AGC TC, for amplifying C-terminus of FCP1 and inserting it into R1/XhoI cut pGEX-4T3. (Tm=60.5)
12)
  fcp-C2b Sal: GAT CGT CGA CCT ACA TGT CAA AGT CCT CCA GG, for amplifying C-terminus of FCP1 and inserting it into R1/XhoI cut pGEX-4T3. (Tm=58.7)
13)
  tat SF1 B.1: GAT CGA ATT CCA TGA GCG ACG AAG GTG GC (begins at ~12; Tm=62.4)
14)
  tat SF1 B.2: GAT CCT CGA GCT TGG TGT TCT GCA ATG G (ends at ~860; Tm=56)
15)
  tatSF1 N-A 5':  TCA GAA TTC GGA CGA AGG TGG CTG TAA ATC, First half of N-terminus preceding the rrm (begins at ~8; Tm=57.3)
16)
  tatSF1 N-A 3':  TAT CTC TCG AGA CGC CAT CCT TGT CGG TGT AA, First half of the N-terminus preceding the rrm (ends at ~556; Tm=66.4)
Row 3 (return to top)
17)
  tatSF1 N-B 5':  CCA GAA TTC AGG CGT TGA TGA AGA TGT TGA TG, second half of the N-terminus preceding the rrm (begins at ~322; Tm=63.5) 
18)
 tatSF1 N-B 3':  TAT CTC TCG AGG CGC TGG ATG CTG GAT TGT CT, second half of the N-terminus preceding the rrm (ends ~780; Tm=67)
19)
  tatSF1 rrm 5':  ATC AGA ATT CAT CCC AGG AGC CCC CAA AAT, amplifies the two rrm (begins ~806; Tm=64.3)
20)
 tatSF1 rrm 3':  TAT CTC TCG AGT AAA ACG ATC GCA GGT AAA TG, amplifies the two rrm (ends ~1700; Tm=57.8)
21)
  tatSF1 N-A.2 5’: CGGGAATTCGGACGAAGGTGGCTGTAAATC; PCR amplifying the N-terminus of dTat-SF1; This is similar to tatSF1 N-A 5' and was made to try to improve EcoR1 cutting at end.
22)
  tatSF1 N-A.2 3’: CTGCGCTCGAGACGCCATCCTTGTCGGTG; PCR amplifying the N-terminus of dTat-SF1; This is similar to tatSF1 N-A 3' and was made to try to improve XhoI cutting at end.
23)
  Sfi1-A: GGC CGC GGC CAT CTA GGC C; insertion of an Sfi1 site into pUAST
24)
  Sfi1-B: TCG AGG CCT AGA TGG CCG C; insertion of an Sfi1 site into pUAST
Row 4 (return to top)
25)
 Gal4-fusion A.1:  CCGGAATTCAAAatgaagctactgtcttctatc; This oligo is for amplifying the Gal4-DBD from pCaSpeR-hs Gal4 1-94 and placing a good Kozak sequence next to the ATG.  It contains an EcoRI site and allows insertion into pCaSpeR-hs
26)
  Gal4-fusion A.2: ccaactggtaatggtagcga; This oligo along with Ga4-fusion A.1 is for amplifying the Gal4-DBD from pCaSpeR-hs Gal4 1-94.  There will be an XbaI site near the end of the fragment.  The PCR product will be cut with EcoR1 and XbaI and inserted into pCaSpeR-hs.
27)
  pGEX5' seq primer: GGG CTG GCA AGC CAC GTT TGG TG
28)
  pGEX3' seq primer: CCG GGA GCT GCA TGT GTC AGA GG
29)
 G-less +35/+10: GGA GTG ATG TGT TTA TTT TGT TTG TT; this oligo is to be used with Larry Benjamins p70 G-less cassette in primer extension assays to monitor polymerase that make it to the promoter proximal region. 
30)
  G-less +344/+318: AAG GAT ATA TGA GAT GAG TAG GGA GTA; this oligo is to be used with Larry's p70 G-less cassette in primer extension assays to monitor polymerase that transcribes well beyond the promoter proximal region.
31)
  p70_+35/+10: TAG CGA CGT GTT CAC TTT GCT TGT TT; this oligo is to be used on p70 and XBX constructs in primte extension assays to monitor polymerase that transcribes into the promoter proximal region.
32)
  p70_+60/+40: CTTGTTTATTTGCTTAGCTTT.  Tm=48.6. +60 to +40 primer for Vivian's analysis of polymerase release. Note, when I ordered this, I mistyped the name and called it p70_+60/+10.
Row 5 (return to top)
33)
  FLAG-NELF-B.1: GCCGAATTCCTGCAGCCCCA; The underlined region matches the 5’ end of the FLAG region in pA5C∆P-Flag-dRD from which we are amplifying the FLAG sequence for the NELF-B subcloning.   Note that this does not encode the FLAG sequence but anneals adjacent the FLAG encoding sequence.
34)
 FLAG-NELF-B.2:  CGGTGTGCTCATTATCATATGCTTGTCATCGTCGTCC; The underlined region matches the 3’ end of the FLAG region in pA5C∆P-Flag-dRD from which we are amplifying the FLAG sequence for the NELF-B subcloning.  The italics matches the 5’ end of NELF-B in Chwen-Huey’s clone that she obtained from Research Genetics.  This oligo provides the connection between the FLAG PCR fragment and the NELF-B coding PCR fragment.
35)
  FLAG-NELF-B.3: ATGATAATGAGCACACCG; The italics matches the 5’ end of NELF-B in Chwen-Huey’s clone that she obtained from Research Genetics.  This is part of the construction of a FLAG-tag NELF-B plasmid
36)
 FLAG-NELF-B.4:  gccagatctCTATTGGACATTATAGTGCG; Lower case contains the BglII site and italics match the 3' end of the NELF-B coding region including the stop codon. This is part of the construction of a FLAG-tag NELF-B plasmid
37)
  NELF-A C-ter.1: CGCggatccCGTGGACTCTCTCTATCGAA; Meghan Smith, For PCR amplifying sequences encoding amino acids 1150-1248 of NELF-A and making pGEX fusion.
38)
  NELF-A C-ter.2: CCGgaattcGCGTATGACCCTTGTGGA; Meghan Smith, For PCR amplifying sequences encoding amino acids 1150-1248 of NELF-A and making pGEX fusion.
39)
  TR-3: CGA CGG GAT CGA TTC CAA TAG GC: this is a fresh preparation of TR-3 for use in ligation mediated PCR analysis of p70 derivatives.
40)
  
Row 6 (return to top)
41)
  RNAi-NELF-B.1: ggcgaattcCCGACGAATTGCATGATAA; Derived from the 5’ end of the translated region of NELF-B with an appended EcoRI site.  This part of the NELF-B RNAi construction.
42)
  RNAi-NELF-B.2: ccgctcgagCCGACGAATTGCATGATAA; Derived from the 5’ end of the translated region of NELF-B with an appended XhoI site. This part of the NELF-B RNAi construction.
43)
  RNAi-NELF-B.3: ggcggccatctaggccCAGCGTGCAATAGTGAACAT; Derived from approximately 800 nucleotides from the AUG with an appended SfiI site. This part of the NELF-B RNAi construction. Use this with RNAi-NELF-B.2
44)
 RNAi-NELF-B.4:  ggcggccaagatggccCAGCGTGCAATAGTGAACAT; Derived from approximately 800 nucleotides from the AUG with an appended SfiI site. This part of the NELF-B RNAi construction.  Use this with RNAi-NELF-B.1
45)
  RNAi-NELF-B.5: ccgtctagaCCGACGAATTGCATGATAA; Derived from the 5’ end of the translated region of NELF-B with an appended XbaI site. This part of the NELF-B RNAi construction.  We switched to an XbaI site because XhoI appeared not to cut the PCR product due to end-sensitivity.  Use this with RNAi-NELF-B.3.
46)
  NELF-A 3'UTR-1: agggacagcagggaaat; For sequencing the junction between the 3’UTR and the putative stop codon for NELF-A
47)
  NELF-A 3'UTR-2: GTGTGCTTGGATGAATGG; For sequencing the junction between the 3’UTR and the putative stop codon for NELF-A
48)
  NELF-A 3'UTR: GGTGTTTTTATGGATGTACGAA; For sequencing the junction between the 3’UTR and the putative stop codon for NELF-A
Row 7 (return to top)
49)
  RNAi-NELF-D.1: ggcgaattcAAAAATGGAAGTGGAATACGA (51 to 71, 5' region near AUG).  Tm for target is 56.  Use with RNAi-NELF-D.2.
50)
  RNAi-NELF-D.2: ggcggccaagatggccGGATAGACTGCCGAACCA (884 to 867, ~800 bp downstream).   Tm for target is 56. 
51)
  RNAi-NELF-D.3: ccgtctagaAAAAATGGAAGTGGAATACGA (5' region near AUG). Tm for target is 56.  Use with RNAi-NELF-D.4
52)
  RNAi-NELF-D.4: ggcggccatctaggccGGATAGACTGCCGAACCA (884 to 867, ~800 bp downstream). Tm for target is 56.
53)
  RNAi-NELF-A.1: ggcgaattcGCGGACAGGCGTAATTC (192 to 208) Tm for target is 58.  Use with RNAi-NELF-A.2.
54)
  RNAi-NELF-A.2: ggcggccaagatggccTAATGCCACCGTCTTTGC (983 to 966) Tm for target is 58.
55)
  RNAi-NELF-A.3: ccgtctagaGCGGACAGGCGTAATTC  (192 to 208) Tm for target is 58.  Use with RNAi-NELF-A.4.
56)
  RNAi-NELF-A.4: ggcggccatctaggccTAATGCCACCGTCTTTGC (983 to 966) Tm for target is 58.
Row 8 (return to top)
57)
  Tailed-1: 5'- GGC AGA TCT TAT CCT CTC CTC ACC TCT C -3'. Zhiqiang will use this to make a tailed template from p70 G-less.
58)
  Tailed-2: 5'- /5Bio/CAC CCC AGG CTT TAC ACT T -3'. Zhiqiang will use this to make a tailed template from p70 G-less.
59)
  Tailed-3: 5'- /5Phos/GAT CAA AAA AAA CCA -3'. Zhiqiang will use this to make a tailed template from p70 G-less.
60)
  fcp pd1: GCT ATG CTA GCG GGC ACG ACG ACA CC; for subcloning phosphatase domain of fcp1 into pET28a.
61)
  fcp pd2: CCT AAA AGC TTA GTC CCC ATG CTT CTC;  for subcloning phosphatase domain of fcp1 into pET28a.
62)
  fcp brca1: GCT TAG CTA GCA TTC GGA GCG AAG TG; for subcloning BRCA1 domain of fcp1 into pET28a.
63)
  fcp brca2: CCA TAA AGC TTA TAC CTT GCG GTC CAG T; for subcloning BRCA1 domain of fcp1 into pET28a.
64)
  

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