Plasmids
ZNF Army
pCAG EV. pCAG EN (CEPKO Lab) was ordered (Addgene plasmid 11160) that contained
multiple cloning sites for subcloning.
pCAG GFP. pCAG GFP (Cepko Lab) was ordered (Addgene plasmid 11150) and used as a
transfection efficiency control as well as a co-transfection plasmid fort FACS applications in the
Tc11 Rescue experiments.
pCAG ZNF91. ZNF91 cDNA sequence was synthesized as a codon-optimized pUC57 insert
flanked by EcoRV sites from Genscript. This insert was EcoRV digested and inserted into the
pCAG EN vector at EcoRV.
pCAG ZNF91 HA. pCAG ZNF91 was used as a starting source to add a C-terminal HA tag via
PCR for Co-IP and western blot analysis.
pCAG ZNF33a. IMAGE clone 8991835 was ordered from ATCC and PCR amplified and
ligated into pENTR/D/TOPO and moved to pCAG-DEST via LR clonase reaction.
pCAG ZNF90. IMAGE clone 9021208 was ordered from ATCC and PCR amplified with a
C-terminal HA tag into pENTR/D/TOPO and moved to pCAG-DEST via LR clonase
reaction.
pCAG ZNF93. cDNA sequence was synthesized as a codon-optimized pUC57 insert flanked by
EcoRV sites from Genscript. This insert was EcoRV digested and inserted into the pCAG EN
vector at EcoRV.
pCAG ZNF93serF HA. ZNF93 Human with the 4 contact residues substituted to Ser at fingers 813 as described(Moore, Choo, & Klug, 2001) thanks to personal communication with Prof. Dave
Segal (UC Davis Genome Center). cDNA sequence was synthesized as a codon-optimized
pUC57 insert flanked by EcoRI/NotI sites from Genscript. This insert was EcoRI/NotI digested
and inserted into the pCAG EN vector at EcoRI/NotI.
pCAG ZNF254. IMAGE clone 5296940 was ordered from ATCC and PCR amplified. This
was ligated into pENTR/D/TOPO and moved to pCAG-DEST with LR reaction.
znf254_ENTR_DG_F-CAC CTG TTA CCA GCA GGT ATT GGA GAT CC
znf254_ENTR_DG_R-GCG CCT CTC TCA GGT TTG TAG TTT C
pCAG ZNF443. IMAGE 5583971 was ordered from ATCC and using restriction digests
was moved form pSPORT6 to pCAG.
pCAG ZNF460. hESC cDNA was used to PCR amplify ZNF460 and this was ligated to
pENTR/D/TOPO. This was moved to pCAG-DEST with LR reaction.
pCAG ZNF486. IMAGE clone 40125819 was ordered from ATCC and PCR amplified with
HA C-terminal primer from pCR4TOPO. This was ligated into pENTR/D/TOPO and
moved to pCAG-DEST with LR reaction.
pCAG ZNF519. IMAGE clone 4806717 was ordered form ATCC and moved from pDNRLIB using restriction enzymes to pCAG.
pCAG ZNF544. IMAGE clone 6199648 in pSPORT6 was ordered from ATCC and using
restriction enzymes was moved to pCAG.
pCAG ZNF587. IMAGE clone 8991953 was ordered through Thermo. This was PCR cloned
from the IMAGE plasmid. The amplicon was ligated to pENTR/D/TOPO and
subsequently moved to pCAG-DEST with LR clonase.
F- caccCCGTGACGGCGACCACTG
R- GCTACTCAGGAGGCTGAGGTAGGAG
pCAG ZNF589. IMAGE clone 100066351 was ordered through Thermo. This was PCR
cloned from the IMAGE plasmid. The amplicon was ligated to pENTR/D/TOPO and
subsequently moved to pCAG-DEST with LR clonase.
pCAG ZNF714. IMAGE clone 4797729 was ordered through ATCC. This was PCR cloned
from pBluescript clone using primers below. This amplicon was ligated to
pENTR/D/TOPO and subsequently moved to pCAG-DEST with LR clonase. This clone
represents unitprot isoform 3 (Q96N38-3).
ZNF714-F caccCTAGAAATGGGCGACCTGAG,
ZNF714-R AGACACCACACCTGGCCTAC
pCAG ZNF721. cDNA sequence was synthesized as a codon-optimized pUC57 insert flanked
from Genscript. This insert was moved to the pCAG EN vector at EcoRV.
Domain deletion analysis of ZNF91
pCAG ZNF91 [1-11]. pCAG ZNF91 was digested with XmaI and NotI and religated resulting in
the removal of the C-terminal 12-36 domains.
pCAG ZNF91 [1-11] HA. pCAG ZNF91 [1-11] was used as a starting source to add a C-terminal
HA tag via PCR and a N-terminal CACC site for ligation into pENTR/D/TOPO (Invitrogen).
This was used in an LR reaction to transfer the insert to pCAG-DEST. This construct was used
for western blot validation of ZNF91 [1-11].
pCAG ZNF91 [1-24]. pCAG ZNF91 was digested with AleI and NotI and religated resulting in
the removal of the C-terminal 25-36 domains.
pCAG ZNF91 [1-30] .pCAG ZNF91 was digested with NsiI and NotI and religated resulting in
the removal of the C-terminal 31-36 domains.
pCAG ZNF91 [1-2; 23-36.]. SacI. present in the gene-optimized synthesized ZNF91
(Genscript) after it was ligated into pCAG.
RECONstruction ZNF91 and ZNF93 Ancestral proteins
pCAG ZNF91hominine HA. ZNF91 representing the LCA of HCG cDNA sequence was
synthesized as a codon-optimized C-terminal HA pUC57 insert flanked by EcoRI and NotI sites
from Genscript. This insert was EcoRI and NotI digested and inserted into the pCAG EN vector
at the EcoRI NotI.
pCAG ZNF91great
ape
HA. ZNF91 representing the LCA of HCGO cDNA sequence was
synthesized as a codon-optimized C-terminal HA pUC57 insert flanked by EcoRI and NotI sites
from Genscript. This insert was EcoRI and NotI digested and inserted into the pCAG EN vector
at the EcoRI NotI.
pCAG ZNF91macaque HA. ZNF91 macaque cDNA sequence was synthesized as a codonoptimized C-terminal HA pUC57 insert flanked by EcoRV sites from Genscript. This insert was
EcoRV digested and inserted into the pCAG EN vector at the EcoRV.
pCAG ZNF93 Human.
pCAG ZNF93great ape. ZNF93 representing the LCA of HCGO cDNA sequence was synthesized
as a codon-optimized C-terminal HA pUC57 insert flanked by EcoRI and NotI sites from
Genscript. This insert was EcoRI and NotI digested and inserted into the pCAG EN vector at the
EcoRI NotI.
pCAG ZNF93macaque. ZNF93 representing the macaque cDNA sequence was synthesized as a
codon-optimized C-terminal HA pUC57 insert flanked by EcoRI and NotI sites from Genscript.
This insert was EcoRI and NotI digested and inserted into the pCAG EN vector at the EcoRI
NotI.
Luciferase Reporter constructs
pGL4cp_SV40. This is a standard promega vector pGL4CP with SV40 minimal promoter
inserted before the firefly luciferase cDNA. This plasmid was a gift from the Privalsky
lab
and
the
sequence
details
can
be
found
here:
http://microbiology.ucdavis.edu/privalsky/sites/default/files/plasmids/pGL4CP_reporters/
pGL4CP-SV40.txt
pGL4CP-SV40-DEST. An rfB cassette (Invitrogen) was introduced at a blunted BglII
upstream of SV40 in pGL4CP-SV40 to give a GATEYWAY compatible destination
vector(Onodera et al., 2012) to screen KAP1+ repeat sequences.
pGL4cp-OCT4Enh-SV40. ~1500bp OCT4 human enhancer region was PCR cloned from
hESC gDNA with primers and ligated upstream of SV40 at the EcoRI blunted site.
OCT4Enh F-CACCCCCTCCACTATGGAACCTGCAC,
OCT4Enh R- AGCTGGCCTTGGCTGAAGTG
>OCT4Enh
CCCTCCACTATGGAACCTGCACATCAGGTTCCTTGCTCCCCTCTCAACCAAAACTC
AGACATCTAATACCACGGTAGGCCCCGTTCTCCCTCCCCCACCTCCCTGGCCCAG
GCCTCCAGCCCTAGGCCCTGGGTGGGGAAAACCAGGGGGTGGGGGGTGTGGAG
AAAAAATATCTGACTTCAGGTTCAAAGAAGCCTGGGAGGGACTGGGGGAAGGGGG
CAGGACAATGGCCTTGGCTGGACAATCCCGGTCCCCAGAGGGGGCAGCTCTAAC
CCTAAACAAGTGCTCAACCCTTGAATGGGCCTGGATGGCTCCCCTGGGGACTGCT
TCCTGCTCCCCAACCCCCCAGTCCCAATCCCCTCACACAGAATCCCCTTCAGAGAC
GCTAAAAGGAGCTCCAGCAACCCCCCTCTGCAATCCCCTCAAAGACTGAGCCTCA
GACGGGCACCAAGGGCCCCCCACAGGGACCTAGGTATCTAGTTCCTCCTTCCTCT
GGGGGACTCAGGCGTCCAGCTTCATCGTGCGTCCCTCCCCGAGCCTGGCAGATT
GAGGGATGTGCTTTGTTTAGTGGGGCTGGCTGGCAGAAAGACGCAGAGGAGGTG
GAGAGTGATTTGTGGAGGCGTGCAGGAAGGCTGCCCTAAGCTCCCCTTCAGGGTC
TGTTTTTCTGGGCCTGGCCTGAGTATCCTGAGGCTCATGCTGCTGGTCTAGTGCTT
GATTCTGTTTGCAAGAGAATAGCCAACGGAATGCCTGTCTGTGAGGGATGATGTTT
GTCTGTCTGCTCCCAAAACTTGATCTCAGTGGAGGGCCTGGGGTAAGTCTGGGGG
CTCCAGAGGGGGCTCTGGGCCAGGGCTCCCCACAGCTTCGAAGGCCAGAAGGCC
AGGTCTGGACTGGGCACGCTGACCTCTGTCGACTTAAGTAAGGCTTCTCATTGCAG
GCTCCAGGCTCAGCCCTGCCTGGGCTTGTCTGCTGAGGTCAGTGGCTCTATCTGC
CTTCTAAGGGGATGGGTGTCCCGTGGCCAGCTGTCTTCATCTTGGTGGCATCCGT
GAGTCTTTTGAGACTTTTCCCCCACTCTTATGTTGCCTCTGTTCGTGTGCCCATCTC
CTGTCTGTGTAGACTTTTTGAGCCTAATTGTATGCGTGCATTTCAATACCTGCCACA
GGTCTGCCGGAAGGTCTACAAGGCAGTGGGGTTGCAGCTGTGTTCACTTCTCGGC
CTTTAACTGCCCAAAAGGCAGGTAGATTATGGGGCCTGGTGGGGGTAGGAGGAAC
ATGCTTCGGAACAGGAGGAGGCCCCTCCCCAGCCATCTCAATCCCCAGGACAGAA
CCATCACGGCACCTTTGTCATGCATCTCTCTGCTGTCTGCCAAGAAGACGGCCTCT
CAGAGGAGGGGGAGGGGCAGGCCTGGGATTTGGCTGGAATCTCCACACCAGTGT
TTCTCAGCTTGCCATCCTCCAGGTTCCCCAAAAGCGCTCTTCCCAAGCCAGTCCAG
AGAGTCCCTGCTGCCCATTTTCCTAGTGGCTCCTAAAACACCTTCCCCAATTTCCC
CACTCAACACCACCCTCTTGTTTTTAGATTATAATTTGTACTGTAGGTGGTGTATTTC
TGGCCTGGGCAAGAGGCCCATTCCCGAGAGGGACGCAGACAAGGGGTGGGTGC
CTGGGTCCCTGGCTGCCTTGTGGCTGGATATGAGCCCAGTCAGGGGTCAGCCTCC
TGCATGCCTAGACTCCTAGCCGGCCCCCTTCTGGGGTGCTCAGGGCTGATGGGA
GGTTGAGGCAGGCTTTCCTTCCTTCTCACTGTCCTGTTATGCCTGAAGGGTAGGTG
GCTTCACTTCAGCCAAGGCCAGCT
pRL-TK. This is a standard promega vector for Renilla with TK minimal promoter
inserted before the renilla luciferase cDNA(Onodera et al., 2012) used as a transfection
efficiency control at a ratio of 10 firefly : 1 renilla to normalize the firefly luciferase
activity in each well (ff/ren).
pGL4cp-SINE-R-SV40. SINE-R domain from SVA_D full length (~XcmI site through
polyA) was synthesized as a gBlock (IDT) and ligated into pENTR/D/TOPO
(Invitrogen). This was moved to pGL4CP-SV40-DEST with the LR GATEWAY
reaction.
pGL4cp-SVA_D-SV40 (full length). Synthesized pUC57-SVA_D (Genescript) was
EcoRV digested and ligated directly to pGL4CP-SV40 at a blunted EcoRI, upstream of
SV40.
> SVA_D-SV40 (full length)
TCTCCCTCTCCCTCTCTTTCCACGGTCTCCCTCTCCCTCTCTTTCCACGGTCTCCCT
CTCCCTCTCTTTCCACGGTCTTCCTCTGATGCCGAGCTGAAGCTGGACTGTACTGC
TGCCATCTCGGCTCACTGCAACCTCCCTGCCTGATTCTCCTGCCTCAGCCTGCCGA
GTGCCTGCGATTGCAGGCGCGCACTGCCACGCCTGACTGGTTTTCGTATTTTTTTG
GTGGAGACAGGGTTTTGCTGTGTTGGCCGGGCTGGTCTCCAGCTCCTAACCGCGA
GTGATCCGCCAGCCTCGGCCTCCCGAAGTGCTGGGATTGCAGACAGAGTCTCGTT
CACTCAGTGCTCAATGGTGCCCAGGCTGGAGTGCAGTGGCCTGATCTCGGCTCGC
TACAACCTCCACCTCCCAGCCGCCTGCCTTGGCCTCCCAAAGTGCCGAGATTGCA
GCCTCTGCCCGGCTGCCACGCCATCTGGGAAGTGAGGAGCGTCTCTGCCTGGCC
GCCCATCGTCTGGGATGTGAGGAGCCCCTCTGCCTGGCTGCCCAGTCTGGGAAG
TGAGGAGCGCCTCTTCCCAGCCGCCATCCCATCTAGGAAGTGAGGAGCGTCTCTG
CCCGGCTGCCCATCGTCTGAGATGTGGGGAGCGCCGCCGCCCTGTCTGGGACTT
GAGGAGCGCCTCTGCCCGGCCGCCCTGTCTGGGATGTGAGGAGCGCCTCTGCCC
GGCCGTGACCCTGTCTGGGAGGTGAGGAGCGTCTCTGCCCGGCCGCCCCGTCTG
AGAAGTGAGGAGCCCCTCCCCCCAGCAGCCGCCCCGTCTGAGAAGTGAGGAGCC
CCTCCGCCCGGCAGCCACCCGATCTGGGAAGTGAGGAGCGTCTCCGCCCGGCAG
CCGCCCCGTCCGGGAGGGAGGTGGGGGGTCAGCCCCCGCCCGGCCAGCCGCC
CCGTCCGGGAGGTGGGGGCGCCTCTGCCCGGCCACCCCTTCTGGGAAGTGAGG
AGCCCCTCTGCCCGGCCACCACCCCGTCTGGAAGGTGTACCCAACAGCTCACTGA
GAACGGGCCATGATGACAATGGCGGTTTTGTGGAATAGAAAAGGGGGAAAGGTGG
GGAAAAGATTGAGAAATCGGATGGTTTCTGTGTCTGTGTAGAAAGAAGTAGACATG
GGAGACTTTTCATTTTGTTCTGTACTAAGAAAAATTCTTCTGCCTTGGGATCCTGTT
GATCTATGACCTTACCCCCAACCCTGTGCTCTCTGAAACATGTGCTGTGTCCACTC
AGGGTTAAATGGATTAAGGGCGGTGCAAGATGTGCTTTGTTAAACAGATGGTCGAA
GGCAGCATGCTCGTTAAGAGTCATCACCACTCCCTAATCTCAAGTACCCAGGGACA
CAAACACTGCGGAAGGCCACAGGGTCCTCTGCCTAGGAAAACCAGAGACCTTTGT
TCACTTGTTTATCTGCTGACCTTCCCTCCACTATTGTCCTATGACCCTGCCAAATCC
CCTCTGCGAGAAACACCCAAGAATGATCAATAAAAAAAAAAAAGAAAAGAA
no hex. pUC57 SVA_D was digested with EcoRV/BglI resulting in ‘ChopD.2’ a
fragment was 1419bp fragment lacking the Hexamer (CCCTCTn)domain. This was
ligated into pGL4CP-SV40, upstream of the SV40 promoter.
no hex/Alu. pUC57 SVA_D was digested with EcoRV/EagI resulting in ‘ChopC.1’ a 787
bp fragment consisting of the 3’ side of SVA and was ligated into pGL4CP-SV40,
upstream of the SV40 promoter.
partial VNTR. pGL4cp-SVA_D-SV40 (full length) was digested with Tth111I which cuts
twice liberating a 401bp fragment, and leaving 1136bp of the SVA_D which was self
ligated as ‘VNTR delta C’
no VNTR. ‘E’. pGL4cp-SVA_D-SV40 (full length) was digested with Tth111I/XcmI
which removes 401bp+321bp fragments, and leaving 813bp of the SVA_D which was
self ligated as ‘VNTR delta D’.
no SINE-R. pUC57 SVA_D was digested with EcoRV/EagI resulting in ‘Chop C.3’ a s
686bp 5’ SVA fragment. This was ligated into pGL4CP-SV40, upstream of the SV40
promoter.
partial Sine-R. pGL4cp-SVA_D-SV40 (full length) was digested with PpuMI/XcmI which
removes a 350bp fragment, and leaving 1187bp of the SVA_D which was self ligated as
VNTR delta A.
Alu-VNTR (1-1.5). pUC57-SVA_D was digested with PfIMI/Tth111I generating a 180bp
Alu domain and VNTR domain containing fragment including 63bp of the VNTR domain
or about 1.5 VNTR repeat units. This was cloned upstream of OCT4Enh at EcoRI blunt in
pGL4cp-OCT4Enh-SV40 as ‘VNTR OCT4Enh F'6’.
VNTR (8.5-15). pUC57-SVA_D was digested with Tth111I/XcmI generating a 323bp
VNTR domain containing fragment or about 6.5 VNTR repeat units. This was cloned
upstream of OCT4Enh at EcoRI blunt in pGL4cp-OCT4Enh-SV40 as ‘VNTR OCT4Enh D3’.
VNTR (1.5-8.5). pUC57-SVA_D was digested with Tth111I/PfIMI generating a 220bp
VNTR domain containing fragment or about 7 VNTR repeat units. This was cloned
upstream of OCT4Enh at EcoRI blunt in pGL4cp-OCT4Enh-SV40 as ‘VNTR OCT4Enh F4’.
Alu-VNTR (1-7.5). pUC57-SVA_D was digested with Tth111I generating a 401bp Alu
and VNTR domain containing fragment or about 7.5 VNTR repeat units. This was cloned
upstream of OCT4Enh at EcoRI blunt in pGL4cp-OCT4Enh-SV40 as ‘VNTR OCT4Enh C6’.
VNTR (1.5-15). pUC57-SVA_D was digested PfImI/XcmI with generating a 544bp VNTR
domain containing fragment or about 13.5 VNTR repeat units. This was cloned upstream
of OCT4Enh at EcoRI blunt in pGL4cp-OCT4Enh-SV40 as ‘VNTR OCT4Enh E2’.
pGL4cp-L1PA4-5’UTR-SV40. Synthesized ~500bp L1PA4 (gBLOCk, IDT) was cloned
into pENTR-D/TOPO (Invitrogen) and transferred by GATEWAY reaction to pGL4CPSV40-DEST.
> L1PA4-5’UTR
GGGGGAGGAGCCAAGATGGCCGAATAGGAACAGCTCCGGTCTACAGCTCCCAGC
GTGAGTGACACAAAAGACGGGTGATTTCTGCATTTCCAACTGAGCTTTGAAGAGAG
TAGTGGTTCTTCCAGCATGCAGCTTGAGATCTGAGAACAGGCAGACTGCCTCCTCA
AGTGGGTCCCTTACCCCCGAGTAGCCTAACTAGGAGGCACCCCCGAGTAGGGGC
AGACTGACACCTCACATGGCAGGGTACTCCTCTGAGACAAAACTTCCAGAGGAAC
GATCAGGCAGCAGCATCTGCAGTTCACCAATATCTGCTGTTCTGCAGCCACTGCTG
CTGATACCCAGGCAAACAGGGTCTGGAGTGGACCTCTAGCAAACTCCAACAGACC
TGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAAACAGAAAGGACATCCA
CACCAAAACCCACCTGTAC
129L1PA4. Synthesized 129bp L1PA4 (gBLOCk, IDT) with flanking sequence from L1HS
at the site of deletion (PvuII sites) was cloned into pENTR-D/TOPO (Invitrogen). This
was next PvuII digested and ligated into pGL4cp-OCT4Enh-SV40 at EcoRI blunt
upstream of OCT4Enh and SV40 promoter.
129scramble. Same as 129L1PA4 except the 129bp element was scrambled before being
syntheized as a gBlock.
51L1PA4. Same as 129L1PA4 except a 51bp region, beginning at the Persikov predicted
18mer/ Zn Finger domains 8-13 was chosen as the anchor point such that 5’-3’ DNA
would be bound by a C-N terminal oriented protein where each finger binds to 3bp.
12951L1PA4. Same as 129L1PA4 except a 51bp region in 51L1PA4 was deleted from the
129bp element at the gBlock synthesis step.
Retrotransposition reporter constructs
L1HS. A gift from the Kazazian lab (JHMI). Thanks to Dustin Hancks for sending
plasmids plasmid map thanks to John Goodier. Hancks description: 99 RPS EGFP- hot
L1, L1-RP (Kimberland et al 1999), cloned into pCEP4 (Invitrogen) lacking the CMV
promoter (Moran et al 1996), L1 is driven by its own promoter in the 5’-UTR, puro
selection, digest with NotI/Bstz17I, two bands 6kb (the L1) and 11 kb backbone + EGFP
retrotransposition indicator cassette, (Ostertag et al 2000 NAR).
L1HS+129bp. L1HS plasmid was Digested with NotI/Bstz17I and ligated to pCAG EN
at NotI/EcoRV. Next, pCAG-L1HS was digested with PvuII, and an insert from the PvuII
fragment from 129L1PA4 was ligated. Finally, pCAG-L1HS-129L1PA4 was digested with
NotI/AleI and ligated back to the pCEP4 based vector that originally contained L1HS
insert. This final plasmid is meant to represent the L1PA4/3 ancestral state.
L1HS+129 scr. Same as L1HS+129bp, except the 129L1PA4 fragment is the 129scramble
insert.
SVA_D and L1PA4 domain analysis was performed with a combination of restriction
enzymes and gBLOCK synthesized fragments.
VNTR-OCT4Enh-pGL4CP-SV40 was created by removing a 545bp, 13.5 repeatcontaining VNTR subunit fragment between PflMI and XcmI. Selected KRAB ZNFs
were obtained from cDNA, synthesized by Genscript, or subcloned from I.M.A.G.E.
vector through ATCC and ligated into pCAG_EN (Cepko Lab, Addgene) either directly
at EcoRI blunt site or through Gateway ENTRY cloning using pCAG_EN with an rfA
cassette integrated at the EcoRI site.