Vector Maps

RETROVIRUS:

To prepare retrovirus, transfect Phoenix cells with retrovector together with CMV-vsvg plasmid for 48hr. Use culture media containing retroviruses to transduce cells such as 3T3-L1 preadipocytes and MEFs.

FOR OVEREXPRESSION:

• pQCXIH (CMV promoter, hygromycin-resistant; CLONTECH, MCS, sequences) - note: MCS (NotI-AgeI-BsiWI-PacI-BamHI) are different from pQCXIP.

• pQCXIP (CMV promoter, puromycin-resistant; CLONTECH, MCS, sequences) - note: MCS (NotI-AgeI-PacI-BamHI- EcoRI) are different from pQCXIH.

• pQCXIP-GFP: a GFP control plasmid

• pBabe-Puro (PL119; low expression level; map) MCS: BamHI - SnaI - EcoRI-SalI. Note: BamHI = BglII and SalI = XhoI. If your construct is in pcDNA3 plasmids using XhoI as 3' end, then you can clone in either BglII-XhoI or EcoRI-XhoI. In many cases, pBabe is better than pQC vectors due to its low expression levels.

• pIRES-GFP RV (PL122; low expression level; map)

• pMSCVhyg (Clontech): Current this vector contains human IRE1a gene (BglII - XhoI); the MCS in pBabe-puro is BamH I-SnaB I-EcoR I-Sal I, whereas that in pMSCVhyg is Bgl II-Xho I-Hpa I. As BamH I/Sal I is compatible to Bgl II/Xho I, we can transfer GOI from pBabe-puro into pMSCVhyg. Expression level should be the same as pBabe. The method of making retrovirus is the same as pBabe (+vsvg in phoenix cells).

FOR RNAi KNOCKDOWN:

References for the pSuper vector, Brummelkamp et al. Science 2002 (pdf): You can make stable knockdown cell lines with these vectors, so it is very useful. Check the design here (jpg). Please note: when using miniprep to screen colonies, please use EcoRI+XhoI (+: 284bp; -: 240bp) - do not use BglII+XhoI because there is no BglII site. You may need to pick 5-8 colonies since the ligation efficiency is pretty low because the oligos are not phosphorylated.

• pSuper.retro.puro (PolIII promoter; puromycin resistance; For more info, please visit Oligoengine, sequence, vector map): this version does not have the Stuffer. Screen miniprep by EcoRI+XhoI (+: 280bp; and -: 240bp; run 1.2% gel).

• pSuper.retro.neo (protocol; sequence = same as the above puro vector, vector map): it has 1kb Stuffer between the two cloning sites BglII and XhoI.

• pSuper.retro.puro.Luciferase RNAi: a negative control.

• pSuper.retro.neo.GFP RNAi: a negative control.

• pSuper.retro.puro.U6 promoter: this is a combination of pBS-U6 adenoviral RNAi construct and pSuper. It can be used for RNAi sequences that have been published using U6 promoter, normally RNAi starts with GGG (map; seq). Sometime it seems better than H1 promoter. Cloning strategy (jpg and doc) is ApaI (blunt) -EcoRI. Miniprep screen strategy is HindIII (350bp) and XhoI (linear).

• For making neo resistant pSuper construct, cut out your fragment from pSuper-puro either H1 or U6) using RI-XhoI --> subclone it into pSuper-neo (RI-XhoI).

• To facilitate subclone into the lentiviral vectors, pSuper (both puro and neo) and pSuper-U6-puro MCS were added two XbaI sites. Check MCS sequences (jpg) here to see where the XbaIs are added.

cDNA FUSIONS (Flag-, HA-, GST-, Gal4- and GFP-)

1. CMV promoter: contains several CRE sites

• pcDNA3-Flag-MCS (PL85) (seq)

• pcDNA3-Myc-MCS

• pcDNA3-His-MCS (derived from PL85) (seq)

• pcDNA3-3xHA-MCS (PL86) (seq)

• pOZ-FH-N/pOZ-FH-C retroviral double tag (up to 6kb; protocol; PL)

• p3x FLAG-CMV-7.1 (PL274 and PL99) (seq)

• pcDNA3-Myc-His A,B,C (PL102~104) (seq)

• pcDNA3-Flag-Gal4(N)-MCS (PL95) (seq)

• pCMX-Gal4(N) (PL94)(seq)>

• pEGFP-N1 (Kan, PL112) (seq)

• pNTAP-B (Kan; PL282) (manual; seq; TAP seq; add 76aa to your protein ~ 7kDa; Note: the frame is already changed to B form - see another map)

• pcDNA3/FKBP DD-MCS (lq434 seq (.doc); FKBP DD seq (.doc); add 10kDa-108aa- to your protein; to make FKBP DD tagged protein)

• pcDNA3.1 (+) (MCS sites; PL106 and 283)

2. SV40 promoter

• pCG SV40 (no tag, PL40) [seq]: only with SV40 promoter, no enhancer.

• pSV40-Flag-MCS (parental vector pCG SV40) [seq]

• pSV40-myc-MCS (parental vector pCG SV40) [seq]

• pSV40-his-MCS (parental vector pCG SV40) [seq]

• pSV40-HA-MCS (parental vector pCG SV40) [seq]

• pMs-Gal4(N)-MCS (lq399~401/PL96~98) (seq): Gal4 fusion

• pRL-SV40 (SV40 promoter-driven Renilla Luciferase)

3. Misc. promoters

• pUB6/V5-His/LacZ (PL2) [seq]: ubiquitin promoter

• pGEX-5X-1 (PL93) [seq]: bacterial promoter driven-GST fusion

COMMON TAG SEQUENCE

Flag:               DYKDDDDK

HA:                 YPYDVPDYA

C-MYC:          EQKLISEEDL

HIS:                HHHHHH

ADENOVIRUS:

• pBS-U6 [image]

• Adtrack-CMV-GFP (Kan)[image]

• Adtrack-CMV (Kan) [image]

• Adeasy [image]

LUCIFERASE:

a. To test the promoter activity:

• pGL3-basic-luc (Promega) () [seq]: cloning vector for promoters

• pFR-luc () [seq]: 5x Gal4 BS-TATATA-luc

• pGL3-gLUC-basic (PL511): cloning vector for promoters; detection of secreted luciferase (gLUC from NEB).

• pTK-Gal4-luc () [seq]: Gal4 DBD binding sites - with much higher basal signals than pFR-luc.