pHTC HaloTag CMV-neo Vector Product Information ... - Promega

pHTC HaloTag ® CMV-neo Vector Features and Circle Map
The following features are present in the pHTC HaloTag ® CMV-neo Vector based on
nucleotide sequence.
CMV intermediate early enhancer/promoter 1–742
Chimeric intron 857–989
T7 RNA polymerase promoter (–17 to +3) 1033–1052
Multiple cloning region 1052–1129
HaloTag ® linker 1124–1168
TEV protease recognition sequence 1139–1159
HaloTag ® coding region 1169–2059
SV40 late polyadenylation signal 2177–2398
SV40 enhancer and early promoter 2497–2915
SV40 minimum origin of replication 2813–2878
Neomycin phophotransferase coding region 2960–3754
Synthetic polyadenylation signal 3818–3866
β-lactamase (Ampr ) coding region 4127–4987
ColE1-derived plasmid origin of replication 5142–5178
ori
Synthetic poly(A)
signal
Amp r
pHTC HaloTag ® CMV-neo
Vector
(6185bp)
Neo r
CMV Enhancer/
Promoter
intron
SV40 Early
Enhancer/
Promoter
T7
TEV
site
SV40 Late
poly(A)
NheI 1053
BmtI 1057
SgfI 1066
PvuI 1066
EcoRI 1074
SacII 1086
EcoRV 1090
XbaI 1092
PspOMI 1101
ApaI 1105
SbfI 1116
EcoICRI 1122
SacI 1124
XhoI 1125
HaloTag ® Open
Reading Frame
Figure 1. pHTC HaloTag ® CMV-neo Vector circle map and sequence reference points.
NheI BmtI
SgfI
PvuI
EcoRI SacII
Usage Information
EcoRV
XbaI
5´– GCT AGC AAA GCG ATC GCT TCC GAA TTC CTA CCG CGG ATA TCT AGA
A S K A I A S E F L P R I S R
PspOMI
ApaI
SbfI
EcoICRI
SacI
XhoI
TTT GGG CCC AAT TCC TGC AGG CGA GCT CTC GAG –3´
F G P N S C R R A L E
Figure 2. pHTC HaloTag ® CMV-neo Vector multiple cloning region sequence and unique
restriction sites. The amino acid sequence corresponds to the correct reading frame for the
HaloTag ® coding region.
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I. Extraction Protocol for the HaloTag ® Vector Card
Materials to be Supplied by User
extraction buffer [10mM Tris (pH 8.0), 20mM NaCl]
E. coli competent cells for transformation (see Section III for suggestions)
1. Cut the vector card on the dotted lines and aseptically remove the filter paper square
containing the dried HaloTag ® Vector DNA (see the QuickTime video for a
demonstration at: www.halotag.com).
2. Transfer the filter paper to a 1.5ml centrifuge tube (e.g., using sterile forceps).
3. Add 30µl of extraction buffer, vortex and briefly centrifuge.
4. Ensure that the filter paper is submerged in the buffer, and incubate for 30 minutes at
room temperature. Vortex sample.
5. Transform 2–10µl of the extracted vector DNA into competent cells, following
manufacturer’s instructions for the competent cells. Store unused DNA at –20°C.
6. Plate the cells on media with Ampr selection, dispensing 2–20% of the
transformation reaction volume.
Note: We recommend working with each vector (N-terminal and C-terminal)
separately to minimize the risk of cross-contamination.
II. Troubleshooting
Symptoms Solutions
Poor transformation Check the competent cells. If necessary, make or purchase
fresh cells.
Check for proper antibiotic selection. Use ampicillin or
carbenicillin to select for the Ampr vector.
Plate a larger volume of the transformation reaction;
if necessary, pellet the transformed cells and resuspend in
100µl of medium. Plate all of the transformed cells.
Dry down the volume of extracted DNA to 5µl and transform.
Note about electroporation
The extraction buffer contains 20mM NaCl, which may be incompatible with
electroporation. Before proceeding with electroporation, ethanol precipitate the vector
DNA from extraction buffer and resuspend in buffer without salt.
III. Related Products
Product Size Cat.#
JM109 Competent Cells, >10 8cfu/µg 5 × 200µl L2001
JM109 Competent Cells, >10 7 cfu/µg 5 × 200µl L1001
HB101 Competent Cells, >10 8cfu/µg 5 × 200µl L2011
HaloTag ® Mammalian Protein Detection and
Purification System 1 each G6795
HaloTag ® Mammalian Pull-Down and Labeling System 24 reactions G6500
HaloCHIP System 20 reactions G9410
Part# GE445
Printed in USA 3/11
Promega Corporation · 2800 Woods Hollow Road·Madison, WI 53711-5399 U.S.A. · Toll Free in the USA 800-356-9526 · Telephone 608-274-4330 · www.promega.com
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