多克隆位點區

Restriction Map and Multiple Cloning Site of pEGFP-N1. (Unique restriction sites are in color or bold.) The Not I site follows the EGFP stop codon. The Xba I site (*) is methylated in the DNA provided by CLONTECH. If you wish to digest the vector with this enzyme, you will need to transform the vector into a dam- host and make fresh DNA.

Description

pEGFP-N1 encodes a red-shifted variant of wild-type GFP (1-3) which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) pEGFP-N1 encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. The MCS in pEGFP-N1 is between the immediate early promoter of CMV (PCMV IE ) and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in the same reading frame as EGFP and there are no intervening stop codons. SV40 polyadenylation signals downstream of the EGFP gene direct proper processing of the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin-resistance cassette (neor ), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex thymidine kinase gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette (Pamp ) expresses kanamycin resistance in E. coli . The pEGFP-N1 backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Use

Fusions to the N-terminus of EGFP retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pEGFP-N1 so that it is in frame with the EGFP coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (7). pEGFP-N1 can also be used simply to express EGFP in a cell line of interest (e.g., as a transfection marker).

Feature Locations

• 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 green fluorescent protein 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
  • GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 871-876
  • 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 EGFP)
• Bacterial promoter expression of Kanr 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 Sites

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

Sequence (text) Sequence (pdf) Restriction Digest (pdf)

全序列：

TAGTTATTAA?TAGTAATCAA?TTACGGGGTC?ATTAGTTCAT?AGCCCATATA?TGGAGTTCCGCGTTACATAA?CTTACGGTAA?ATGGCCCGCC?TGGCTGACCG?CCCAACGACC?CCCGCCCATTGACGTCAATA?ATGACGTATG?TTCCCATAGT?AACGCCAATA?GGGACTTTCC?ATTGACGTCAATGGGTGGAG?TATTTACGGT?AAACTGCCCA?CTTGGCAGTA?CATCAAGTGT?ATCATATGCCAAGTACGCCC?CCTATTGACG?TCAATGACGG?TAAATGGCCC?GCCTGGCATT?ATGCCCAGTACATGACCTTA?TGGGACTTTC?CTACTTGGCA?GTACATCTAC?GTATTAGTCA?TCGCTATTACCATGGTGATG?CGGTTTTGGC?AGTACATCAA?TGGGCGTGGA?TAGCGGTTTG?ACTCACGGGGATTTCCAAGT?CTCCACCCCA?TTGACGTCAA?TGGGAGTTTG?TTTTGGCACC?AAAATCAACGGGACTTTCCA?AAATGTCGTA?ACAACTCCGC?CCCATTGACG?CAAATGGGCG?GTAGGCGTGTACGGTGGGAG?GTCTATATAA?GCAGAGCTGG?TTTAGTGAAC?CGTCAGATCC?GCTAGCGCTACCGGACTCAG?ATCTCGAGCT?CAAGCTTCGA?ATTCTGCAGT?CGACGGTACC?GCGGGCCCGGGATCCACCGG?TCGCCACCAT?GGTGAGCAAG?GGCGAGGAGC?TGTTCACCGG?GGTGGTGCCCATCCTGGTCG?AGCTGGACGG?CGACGTAAAC?GGCCACAAGT?TCAGCGTGTC?CGGCGAGGGCGAGGGCGATG?CCACCTACGG?CAAGCTGACC?CTGAAGTTCA?TCTGCACCAC?CGGCAAGCTGCCCGTGCCCT?GGCCCACCCT?CGTGACCACC?CTGACCTACG?GCGTGCAGTG?CTTCAGCCGCTACCCCGACC?ACATGAAGCA?GCACGACTTC?TTCAAGTCCG?CCATGCCCGA?AGGCTACGTCCAGGAGCGCA?CCATCTTCTT?CAAGGACGAC?GGCAACTACA?AGACCCGCGC?CGAGGTGAAGTTCGAGGGCG?ACACCCTGGT?GAACCGCATC?GAGCTGAAGG?GCATCGACTT?CAAGGAGGACGGCAACATCC?TGGGGCACAA?GCTGGAGTAC?AACTACAACA?GCCACAACGT?CTATATCATGGCCGACAAGC?AGAAGAACGG?CATCAAGGTG?AACTTCAAGA?TCCGCCACAA?CATCGAGGACGGCAGCGTGC?AGCTCGCCGA?CCACTACCAG?CAGAACACCC?CCATCGGCGA?CGGCCCCGTGCTGCTGCCCG?ACAACCACTA?CCTGAGCACC?CAGTCCGCCC?TGAGCAAAGA?CCCCAACGAGAAGCGCGATC?ACATGGTCCT?GCTGGAGTTC?GTGACCGCCG?CCGGGATCAC?TCTCGGCATGGACGAGCTGT?ACAAGTAAAG?CGGCCGCGAC?TCTAGATCAT?AATCAGCCAT?ACCACATTTGTAGAGGTTTT?ACTTGCTTTA?AAAAACCTCC?CACACCTCCC?CCTGAACCTG?AAACATAAAATGAATGCAAT?TGTTGTTGTT?AACTTGTTTA?TTGCAGCTTA?TAATGGTTAC?AAATAAAGCAATAGCATCAC?AAATTTCACA?AATAAAGCAT?TTTTTTCACT?GCATTCTAGT?TGTGGTTTGTCCAAACTCAT?CAATGTATCT?TAAGGCGTAA?ATTGTAAGCG?TTAATATTTT?GTTAAAATTCGCGTTAAATT?TTTGTTAAAT?CAGCTCATTT?TTTAACCAAT?AGGCCGAAAT?CGGCAAAATCCCTTATAAAT?CAAAAGAATA?GACCGAGATA?GGGTTGAGTG?TTGTTCCAGT?TTGGAACAAGAGTCCACTAT?TAAAGAACGT?GGACTCCAAC?GTCAAAGGGC?GAAAAACCGT?CTATCAGGGCGATGGCCCAC?TACGTGAACC?ATCACCCTAA?TCAAGTTTTT?TGGGGTCGAG?GTGCCGTAAAGCACTAAATC?GGAACCCTAA?AGGGAGCCCC?CGATTTAGAG?CTTGACGGGG?AAAGCCGGCGAACGTGGCGA?GAAAGGAAGG?GAAGAAAGCG?AAAGGAGCGG?GCGCTAGGGC?GCTGGCAAGTGTAGCGGTCA?CGCTGCGCGT?AACCACCACA?CCCGCCGCGC?TTAATGCGCC?GCTACAGGGCGCGTCAGGTG?GCACTTTTCG?GGGAAATGTG?CGCGGAACCC?CTATTTGTTT?ATTTTTCTAAATACATTCAA?ATATGTATCC?GCTCATGAGA?CAATAACCCT?GATAAATGCT?TCAATAATATTGAAAAAGGA?AGAGTCCTGA?GGCGGAAAGA?ACCAGCTGTG?GAATGTGTGT?CAGTTAGGGTGTGGAAAGTC?CCCAGGCTCC?CCAGCAGGCA?GAAGTATGCA?AAGCATGCAT?CTCAATTAGTCAGCAACCAG?GTGTGGAAAG?TCCCCAGGCT?CCCCAGCAGG?CAGAAGTATG?CAAAGCATGCATCTCAATTA?GTCAGCAACC?ATAGTCCCGC?CCCTAACTCC?GCCCATCCCG?CCCCTAACTCCGCCCAGTTC?CGCCCATTCT?CCGCCCCATG?GCTGACTAAT?TTTTTTTATT?TATGCAGAGGCCGAGGCCGC?CTCGGCCTCT?GAGCTATTCC?AGAAGTAGTG?AGGAGGCTTT?TTTGGAGGCCTAGGCTTTTG?CAAAGATCGA?TCAAGAGACA?GGATGAGGAT?CGTTTCGCAT?GATTGAACAAGATGGATTGC?ACGCAGGTTC?TCCGGCCGCT?TGGGTGGAGA?GGCTATTCGG?CTATGACTGGGCACAACAGA?CAATCGGCTG?CTCTGATGCC?GCCGTGTTCC?GGCTGTCAGC?GCAGGGGCGCCCGGTTCTTT?TTGTCAAGAC?CGACCTGTCC?GGTGCCCTGA?ATGAACTGCA?AGACGAGGCAGCGCGGCTAT?CGTGGCTGGC?CACGACGGGC?GTTCCTTGCG?CAGCTGTGCT?CGACGTTGTCACTGAAGCGG?GAAGGGACTG?GCTGCTATTG?GGCGAAGTGC?CGGGGCAGGA?TCTCCTGTCATCTCACCTTG?CTCCTGCCGA?GAAAGTATCC?ATCATGGCTG?ATGCAATGCG?GCGGCTGCATACGCTTGATC?CGGCTACCTG?CCCATTCGAC?CACCAAGCGA?AACATCGCAT?CGAGCGAGCACGTACTCGGA?TGGAAGCCGG?TCTTGTCGAT?CAGGATGATC?TGGACGAAGA?GCATCAGGGGCTCGCGCCAG?CCGAACTGTT?CGCCAGGCTC?AAGGCGAGCA?TGCCCGACGG?CGAGGATCTCGTCGTGACCC?ATGGCGATGC?CTGCTTGCCG?AATATCATGG?TGGAAAATGG?CCGCTTTTCTGGATTCATCG?ACTGTGGCCG?GCTGGGTGTG?GCGGACCGCT?ATCAGGACAT?AGCGTTGGCTACCCGTGATA?TTGCTGAAGA?GCTTGGCGGC?GAATGGGCTG?ACCGCTTCCT?CGTGCTTTACGGTATCGCCG?CTCCCGATTC?GCAGCGCATC?GCCTTCTATC?GCCTTCTTGA?CGAGTTCTTCTGAGCGGGAC?TCTGGGGTTC?GAAATGACCG?ACCAAGCGAC?GCCCAACCTG?CCATCACGAGATTTCGATTC?CACCGCCGCC?TTCTATGAAA?GGTTGGGCTT?CGGAATCGTT?TTCCGGGACGCCGGCTGGAT?GATCCTCCAG?CGCGGGGATC?TCATGCTGGA?GTTCTTCGCC?CACCCTAGGGGGAGGCTAAC?TGAAACACGG?AAGGAGACAA?TACCGGAAGG?AACCCGCGCT?ATGACGGCAATAAAAAGACA?GAATAAAACG?CACGGTGTTG?GGTCGTTTGT?TCATAAACGC?GGGGTTCGGTCCCAGGGCTG?GCACTCTGTC?GATACCCCAC?CGAGACCCCA?TTGGGGCCAA?TACGCCCGCGTTTCTTCCTT?TTCCCCACCC?CACCCCCCAA?GTTCGGGTGA?AGGCCCAGGG?CTCGCAGCCAACGTCGGGGC?GGCAGGCCCT?GCCATAGCCT?CAGGTTACTC?ATATATACTT?TAGATTGATTTAAAACTTCA?TTTTTAATTT?AAAAGGATCT?AGGTGAAGAT?CCTTTTTGAT?AATCTCATGACCAAAATCCC?TTAACGTGAG?TTTTCGTTCC?ACTGAGCGTC?AGACCCCGTA?GAAAAGATCAAAGGATCTTC?TTGAGATCCT?TTTTTTCTGC?GCGTAATCTG?CTGCTTGCAA?ACAAAAAAACCACCGCTACC?AGCGGTGGTT?TGTTTGCCGG?ATCAAGAGCT?ACCAACTCTT?TTTCCGAAGGTAACTGGCTT?CAGCAGAGCG?CAGATACCAA?ATACTGTCCT?TCTAGTGTAG?CCGTAGTTAGGCCACCACTT?CAAGAACTCT?GTAGCACCGC?CTACATACCT?CGCTCTGCTA?ATCCTGTTACCAGTGGCTGC?TGCCAGTGGC?GATAAGTCGT?GTCTTACCGG?GTTGGACTCA?AGACGATAGTTACCGGATAA?GGCGCAGCGG?TCGGGCTGAA?CGGGGGGTTC?GTGCACACAG?CCCAGCTTGGAGCGAACGAC?CTACACCGAA?CTGAGATACC?TACAGCGTGA?GCTATGAGAA?AGCGCCACGCTTCCCGAAGG?GAGAAAGGCG?GACAGGTATC?CGGTAAGCGG?CAGGGTCGGA?ACAGGAGAGCGCACGAGGGA?GCTTCCAGGG?GGAAACGCCT?GGTATCTTTA?TAGTCCTGTC?GGGTTTCGCCACCTCTGACT?TGAGCGTCGA?TTTTTGTGAT?GCTCGTCAGG?GGGGCGGAGC?CTATGGAAAAACGCCAGCAA?CGCGGCCTTT?TTACGGTTCC?TGGCCTTTTG?CTGGCCTTTT?GCTCACATGTTCTTTCCTGC?GTTATCCCCT?GATTCTGTGG?ATAACCGTAT?TACCGCCATG?CAT