pEYFP-N1

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pEYFP-N1 Vector Information PT3192-5Catalog #6006-1

Restriction Map and Multiple Cloning Site (MCS) of pEYFP-N1. Unique restriction sites are in bold. The Xba I site (*) is

methylated in the DNA provided by BD Biosciences Clontech. If you wish to digest this vector with this enzyme, you will need to transform the vector into a dam – host and make fresh DNA.

Description:

pEYFP-N1 encodes an enhanced yellow-green variant of the Aequorea victoria green fluorescent protein (GFP). The EYFP gene contains four amino acid substitutions previously published as GFP-10C (1). The fluorescence excitation maximum of EYFP is 513 nm, and the emission spectrum has a peak at 527 nm (in the yellow-green region). When excited at 513 nm, the E m of EYFP is 36,500 cm –1M –1 and the fluorescence quantum yield is 0.63 (1), resulting in a bright fluorescent signal.The fluorescence level observed from EYFP is roughly equivalent to that from EGFP.

In addition to the chromophore mutations, EYFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (2). Furthermore, upstream sequences flanking EYFP have been converted to a Kozak consensus translation initiation site (3). These changes increase the translational efficiency of the EYFP mRNA and consequently the expression of EYFP in mammalian and plant cells.

The MCS in pEYFP-N1 is between the immediate early promoter of CMV (P CMV IE ) and the EYFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EYFP if they are in the same reading frame as EYFP and there are no intervening stop codons. The inserted gene should include an initiating ATG codon. EYFP with N-terminal fusion moieties retains the fluorescent properties of the native protein and thus can be used to localize fusion proteins in vivo .

The vector contains an SV40 origin of replication and a neomycin resistance (Neo r ) gene for selection (using G418) in mammalian cells. A bacterial promoter upstream of this cassette (P ) expresses kanamycin resistance in E. coli . The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

The recombinant EYFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (4). pEYFP-N1 can also be used simply to express EYFP in a cell line of interest (e.g., as a transfection marker). EGFP, EYFP, and EBFP variants can be used independently or in combination for flow cytometry analysis.

I (1402) (1640)

Eco (3856)

(2579)

Ase I

I* (1412)G I (1389)

I (888)

G CTA GCG CTA CCG GAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG ACG GTA CCG CGG GCC CGG GAT CCA CCG GTC GCC ACC 591?

611?

601?

621?

631?

641?

661?

651?

671?

Hin d III Xho I Apa I Bsp 120 I Kpn I Asp 718 I Bam H I EYFP

Age I Xma I Sma I

Sac II Sal I Acc I Sac I Ecl 136 II Eco R I Pst I Eco 47 III

Bgl II Nhe I (PR29945; published 03 Octobers 2002)

Location of features:

?Human cytomegalovirus (CMV) immediate early promoter: 1–589

Enhancer region: 59–465

TATA box: 554–560

Transcription start point: 583

C→G mutation to remove Sac I site: 569

?MCS: 591–671

?Enhanced yellow fluorescent protein (EYFP) gene

Kozak consensus translation initiation site: 672–682

Start codon (ATG): 679–681; stop codon: 1396–1398

Insertion of Val at position 2: 682–684

GFP-10C mutations (Ser-65 to Gly: 874–876; Val-68 to Leu: 883–885; Ser-72 to Ala: 895–897; Thr-203 to Tyr: 1288–1290)

His-231 to Leu mutation (A→T): 1373

?SV40 early mRNA polyadenylation signal

Polyadenylation signals: 1552–1557 & 1581–1586

mRNA 3' ends: 1590 & 1602

?f1 single-strand DNA origin: 1649–2104

(Packages the noncoding strand of EYFP.)

?Bacterial promoter for expression of Kan r gene:

–35 region: 2166–2171; –10 region: 2189–2194

Transcription start point: 2201

?SV40 origin of replication: 2445–2580

?SV40 early promoter

Enhancer (72-bp tandem repeats): 2278–2349 & 2350–2421

21-bp repeats: 2425–2445, 2446–2466, & 2468–2488

Early promoter element: 2501–2507

Major transcription start points: 2497, 2535, 2541 & 2546

?Kanamycin/neomycin resistance gene

Neomycin phosphotransferase coding sequences:

Start codon (ATG): 2629–2631; stop codon: 3421–3423

G→A mutation to remove Pst I site: 2811

C→A (Arg to Ser) mutation to remove Bss H II site: 3157

?Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 3659–3664 & 3672–3677

?pUC plasmid replication origin: 4008–4651

Primer Locations:

?EGFP-N Sequencing Primer (#6479-1): 745–724

?EGFP-C Sequencing Primer (#6478-1): 1332–1353

Propagation in E. coli:

?Suitable host strains: DH5α, HB101, and other general-purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM101 or XL1-Blue.

?Selectable marker: plasmid confers resistance to kanamycin (30 μg/ml) in E. coli hosts.

? E. coli replication origin: pUC

?Copy number: ~500

?Plasmid incompatibility group: pMB1/ColE1

References:

1.Orm?, M. et al. (1996) Science273:1392–1395.

2.Haas, J., et al. (1996) Curr. Biol.6:315–324.

3.Kozak, M. (1987) Nucleic Acids Res.15:8125–8148.

4.Gorman, C. (1985) In DNA cloning: a practical approach, vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.

Note: The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by BD Biosciences Clontech. This vector has not been completely sequenced.

Notice to Purchaser

Use of BD Biosciences Clontech’s Living Colors? products containing DNA sequences coding for mutant Aequorea victoria green fluorescent protein (GFP) variants or proteins thereof requires a license from Amersham Biosciences under U.S. Patent Nos. 5,625,048; 5,777,079; 6,054,321 and other pending U.S. and foreign patent applications. In addition, certain BD Biosciences Clontech products are made under U.S. Patent No. 5,804,387 licensed from Stanford University.

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