pGFPuv Vector Information
PT3055-5
GenBank Accession #U62636
Cat. No. 632312
Sap I
5' MCS
(3330)
AlwN I
P
(2804)
(234–281)
Nco I (455)
BsrG I (565)
Mlu I (611)
lac
pUC
ori
GFPuv
Xho I (710)
pGFPuv
3.3 kb
BstB I (911)
Sac I (996)
3' MCS
Ampr
(1007–1091)
Aat II (1406)
Pvu I
(1956)
5' MCS
230
•
lacZ
250
•
240
•
260
•
270
•
280
•
GFPuv
ATG ACC ATG ATT ACG CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG GTA GAA AAA ATG AGT
Hind III
Xba I
Sph I
Xma I Kpn I Age I
Pst I
Sma I Asp718 I
3' MCS
GFPuv
STOP
1010
•
1020
•
1030
•
1040
•
1050
•
1060
•
1070
•
1080
•
1090
•
TAA TGAATTCCAACTGAGCGCCGGTCGCTACCATTACCAACTTGTCTGGTGTCAAAAATAATAGGCCTACTAGTCGGCCGTACGGGCCC
Bsp10 I
Stu I Spe I Eag I
EcoR I
BsiW I Apa I
Restriction Map and Multiple Cloning Site (MCS) of pGFPuv Vector. Unique restriction sites are in bold.
Description:
pGFPuv carries the “cycle 3” variant of GFP described by Crameri et al. (1). This gene was
cloned between the two MCSs of the pUC19 derivative pPD16.43 (2). The GFPuv gene can
be easily excised from pGFPuv. Alternatively, the GFPuv coding sequence can be amplified by
PCR. The GFPuv gene was inserted in frame with the lacZ initiation codon from pUC19 so that a
β-galactosidase-GFPuv fusion protein is expressed from the lac promoter in E. coli . Note, however,
that if you excise the GFPuv coding sequence using a restriction site in the 5' MCS, the resulting
fragment will encode the native (i.e., non-fusion) GFPuv protein. The pUC backbone of pGFPuv
provides a high copy number origin of replication and ampicillin resistance gene for propagation in
E. coli.
(PR29966; published 03 October 2002)
pGFPuv
Vector Information
Location of features:
• lac promoter: 95–178
CAP binding site: 111–124
–35 region: 143–148; –10 region: 167–172
Transcription start point: 179
lac operator: 179–199
• lacZ-GFPuv fusion protein expressed in E. coli
Ribosome binding site: 206–209
Start codon (ATG): 217–219; Stop codon: 1003–1005
• 5' MCS: 234–281
• GFPuv gene
Start codon (ATG): 289–291; Stop codon: 1003–1005
GFP chromophore: 481–489
wt GFP cDNA sequences (3): 289–454
Synthetic GFP gene with "cycle 3" mutations from pBAD-GFPuv (1): 455–1007
Cycle 3 mutation F99S (T→C): 584
Cycle 3 mutation M153T (T→C): 7Cycle 3
Cycle 3 mutation V163A (T→C): 776
Cycle 3 silent mutation in L137 (T→C): 699
Cycle 3 silent mutation in T225 (A→T): 963
Q80R mutation (A→G) (4): 527
Arg codons optimized for E. coli: R73 (AgA→CgT): 505–507, R96 (AgA→CgC): 574–576,
R12(AgA→CgT): 652–654, R168 (AgA→CgC): 790–792, R1215 (AgA→CgT): 931–933
Silent mutations (CccA→TccG) creating BspE I site: 510 & 513
Silent mutation (A→G) creating Mlu I site: 612
Silent mutations (TtGgaA→CtCgaG) creating Xho I site: 709, 711 & 714
Silent mutations (AG→TC) creating BamH I site: 811–812
Silent mutation (C→G) creating Sal I site: 894
Silent mutations (ActA→GctC) creating Sac I site: 993 & 996
Silent mutation in S72 (A→C): 504 • 3' MCS: 107–1091
• Ampicilin resistance gene
Promoter: –35 region: 1467–1472; –10 region: 1490–1495
Transcription start point: 1502
Ribosome binding site: 1525–1529
β-lactamase coding sequences:
Start codon (ATG): 1537–1539; Stop codon: 2395–2397
β-lactamase signal peptide: 1537–1605
β-lactamase mature protein: 1606–2394
• pUC plasmid replication origin: 2545–3188
Primer Location:
• GFP-N Sequencing Primer: 331–352
(Note: The GFP-C Sequencing Primer cannot be used with pGFPuv.)
Propagation in E. coli:
• Recommended host strain: JM109 or DH5α
• Selectable marker: plasmid confers resistance to ampicillin (100 µg/ml) on E. coli hosts.
• E. coli replication origin: pUC
• Copy number: ≈500
• Plasmid incompatibility group: pMB1/ColE1
References:
1.
2.
3.
4.
Crameri, A., et al. (1996) Nature Biotechnol. 14:315–319.
Fire, A., et al. (1990) Gene 93:189–198.
Prasher, D. C., et al. (1992) Gene 111:229–233.
Chalfie, M., et al. (1994) Science 263:802–805.
Clontech Laboratories, Inc.
www.clontech.com
Protocol No. PT3055-5
Version No. PR29966
Fri, Jul 12, 1996
Digest 1 of pGFPuv: 3337 bases (circular)
Page 1
AseI
PvuII
|
|
AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTT
TCGCGGGTTATGCGTTTGGCGGAGAGGGGCGCGCAACCGGCTAAGTAATTACGTCGACCGTGCTGTCCAA
10
20
30
40
50
60
70
AseI
|
TCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAG
AGGGCTGACCTTTCGCCCGTCACTCGCGTTGCGTTAATTACACTCAATCGAGTGAGTAATCCGTGGGGTC
80
90
100
110
120
130
140
BsrBI
|
GCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAA
CGAAATGTGAAATACGAAGGCCGAGCATACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTT
150
160
170
180
190
200
210
Sal I
Asp718 I
BspMI
PstI
XbaI
XmaI
KpnI
HindIII
SphI Sse8387 I
BamHI SmaI AgeI
|
|
|
| |
|
|
| | | ||
ACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGG
TGTCGATACTGGTACTAATGCGGTTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATGGCC
220
230
240
250
260
270
280
BpmI
|
TAGAAAAAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGA
ATCTTTTTTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACT
290
300
310
320
330
340
350
TGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT
ACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCACTTCCACTACGTTGTATGCCTTTTGAATGGGAA
360
370
380
390
400
410
420
NcoI MscI
|
|
AAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTG
TTTAAATAAACGTGATGACCTTTTGATGGACAAGGTACCGGTTGTGAACAGTGATGAAAGAGAATACCAC
430
440
450
460
470
480
490
BspEI
NdeI
|
|
TTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGG
AAGTTACGAAAAGGGCAATAGGCCTAGTATACTTTGCCGTACTGAAAAAGTTCTCACGGTACGGGCTTCC
500
510
520
530
540
550
560
BsrGI
MluI
|
|
TTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTT
AATACATGTCCTTGCGTGATATAGAAAGTTTCTACTGCCCTTGATGTTCTGCGCACGACTTCAGTTCAAA
570
580
590
600
610
620
630