The vector pGRFP was developed to report the integration of cloned inserts by shifting the ratio of green to red fluorescence. In the native vector, a fusion protein composed of GFP and rapidly maturing DsRed-T3 fluorescent protein is expressed.
| 载体基本信息 | |||
| 载体名称 | pGRFP | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 3441 bp | 载体类型 | Dual fluorescent protein cloning vector |
| 载体来源 | Choe J, Guo HH, van den Engh G. | ||
pGRFP 载体载体图谱
pGRFP 载体载体序列
LOCUS Exported 3441 bp ds-DNA circular SYN 18-DEC-2018
DEFINITION Dual fluorescent protein cloning vector pGRFP, complete sequence.
ACCESSION AY916793
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3441)
AUTHORS Choe J, Guo HH, van den Engh G.
TITLE A dual-fluorescence reporter system for high-throughput clone
characterization and selection by cell sorting
JOURNAL Nucleic Acids Res. 33 (5), E49 (2005)
PUBMED 15767274
REFERENCE 2 (bases 1 to 3441)
AUTHORS Choe J, Guo HH, van den Engh G.
TITLE Direct Submission
JOURNAL Submitted (01-FEB-2005) Laboratory of Ger van den Engh, Institute
for Systems Biology, 1441 N 34th St, Seattle, WA 98103-8904, USA
REFERENCE 3 (bases 1 to 3441)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..3441
/organism="Dual fluorescent protein cloning vector pGRFP"
/mol_type="other DNA"
/db_xref="taxon:314306"
protein_bind 49..70
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 85..115
/label=lac promoter
/note="promoter for the E. coli lac operon"
RBS 126..134
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3 end of the 16S rRNA (Chen et al., 1994)"
CDS 140..1630
/codon_start=1
/gene="GRFP"
/product="GFP-DsRed fusion protein"
/label=GRFP
/note="fusion protein containing GFPmut3.1 and DsRed-T3
with an intervening linker region"
/protein_id="AAX13969.1"
/translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYKSGSGSGCKTTASSAAADISVDGHSCFLGSGSGSMASSEDVIKE
FMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGS
KVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGCFIYKVKFIGVN
FPSDGPVMQKKTMGWEPSTERLYPRDGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPV
QLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHHLFL"
gene 140..1630
/gene="GRFP"
/label=GRFP
misc_feature 140..853
/gene="GRFP"
/note="cycle 3 mutant of GFP; derived initially from
pGFPmut3.1; Region: green fluorescent protein mutant 3.1"
CDS 140..853
/codon_start=1
/product="Aequoria victoria green fluorescent protein"
/label=GFP
/translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
misc_feature 854..952
/gene="GRFP"
/note="contains G,S residues for flexibility on each side,
M13 forward and reverse primer sites, and NotI, EcoRV, SalI
cloning sites; Region: linker"
primer_bind 873..889
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(918..934)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
CDS 953..1630
/codon_start=1
/product="rapidly maturing tetrameric variant of DsRed
fluorescent protein (Bevis and Glick, 2002)"
/label=DsRed-Express
/note="mammalian codon-optimized"
/translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEPSTERLYPRDGVLKGEIHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
LFL"
misc_feature 953..1627
/gene="GRFP"
/note="faster folding mutant of discosoma red fluorescent
protein; derived from pDsRed-T3; silent mutations have been
introduced to change restriction sites; Region: discosoma
red fluorescent protein T3 mutant"
promoter 1673..1777
/gene="bla"
/label=AmpR promoter
CDS 1778..2638
/codon_start=1
/product="beta-lactamase"
/function="ampicillin resistance"
/label=beta-lactamase
/protein_id="AAX13968.1"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2809..3397
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
