pAcGFP1-1

pAcGFP1-1

pAcGFP1-1载体基本信息：

pAcGFP1-1载体质粒图谱和多克隆位点信息：

pAcGFP1-1载体描述：

pAcGFP1-1 encodes the green fluorescent protein AcGFP1, a derivative of AcGFP from Aequorea coerulescens. AcGFP1 has been optimized for brighter fluorescence. (Excitation maximum = 475 nm; emission maximum = 505 nm.) The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1). pAcGFP1-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of AcGFP1 have been converted to a Kozak consensus translation initiation site (2) to enhance translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.

AcGFP1 can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pAcGFP1-1 MCS upstream from the AcGFP1 coding sequence. Without addition of a functional promoter, this vector will not express AcGFP1. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard method. If required, stable transfectants can be selected using G418 (3).

pAcGFP1-1载体含有以下元件：

MCS: 12–83

 Aequorea coerulescens green fluorescent protein (AcGFP1) gene

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 814-816

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 969–974 & 998–1003

mRNA 3' ends: 1007 & 1019

 f1 single-strand DNA origin: 1066–1521

(Packages noncoding strand of AcGFP1-1.)

 Ampicillin resistance (β-lactamase) promoter

–35 region: 1583–1588; –10 region: 1606–1611

Transcription start point: 1618

 SV40 origin of replication: 1862–1997

 SV40 early promoter

Enhancer (72-bp tandem repeats): 1693–1766 & 1767–1838

21-bp repeats: 1842–1862, 1863–1883 & 1885–1905

Early promoter element: 1918–1924

Major transcription start points: 1914, 1952, 1958 & 1963

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 2046–2048; stop codon: 2838–2840

G→A mutation to remove Pst I site: 2228

C→A (Arg→Ser) mutation to remove BssH II site: 2574

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

Polyadenylation signals: 3076–3081 & 3089–3094

 pUC plasmid replication origin: 3425–4068

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 JM109 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~500

pAcGFP1-1载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG TGAGCAAGGG CGCCGAGCTG

  121 TTCACCGGCA TCGTGCCCAT CCTGATCGAG CTGAATGGCG ATGTGAATGG CCACAAGTTC

  181 AGCGTGAGCG GCGAGGGCGA GGGCGATGCC ACCTACGGCA AGCTGACCCT GAAGTTCATC

  241 TGCACCACCG GCAAGCTGCC TGTGCCCTGG CCCACCCTGG TGACCACCCT GAGCTACGGC

  301 GTGCAGTGCT TCTCACGCTA CCCCGATCAC ATGAAGCAGC ACGACTTCTT CAAGAGCGCC

  361 ATGCCTGAGG GCTACATCCA GGAGCGCACC ATCTTCTTCG AGGATGACGG CAACTACAAG

  421 TCGCGCGCCG AGGTGAAGTT CGAGGGCGAT ACCCTGGTGA ATCGCATCGA GCTGACCGGC

  481 ACCGATTTCA AGGAGGATGG CAACATCCTG GGCAATAAGA TGGAGTACAA CTACAACGCC

  541 CACAATGTGT ACATCATGAC CGACAAGGCC AAGAATGGCA TCAAGGTGAA CTTCAAGATC

  601 CGCCACAACA TCGAGGATGG CAGCGTGCAG CTGGCCGACC ACTACCAGCA GAATACCCCC

  661 ATCGGCGATG GCCCTGTGCT GCTGCCCGAT AACCACTACC TGTCCACCCA GAGCGCCCTG

  721 TCCAAGGACC CCAACGAGAA GCGCGATCAC ATGATCTACT TCGGCTTCGT GACCGCCGCC

  781 GCCATCACCC ACGGCATGGA TGAGCTGTAC AAGTGAGCGG CCGCGACTCT AGATCATAAT

  841 CAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT

  901 GAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA

  961 TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA

 1021 TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA

 1081 ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG

 1141 CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG

 1201 TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA

 1261 AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG

 1321 GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT

 1381 GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG

 1441 CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA

 1501 ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA

 1561 TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT

 1621 AAATGCTTCA ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA

 1681 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG

 1741 CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG

 1801 AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC

 1861 CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT

 1921 TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG

 1981 AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT

 2041 TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC

 2101 TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC

 2161 TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG

 2221 AACTGCAAGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG

 2281 CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG

 2341 GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG

 2401 CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC

 2461 ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG

 2521 ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC

 2581 CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG

 2641 AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC

 2701 AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC

 2761 GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC

 2821 TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC

 2881 CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG

 2941 AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT

 3001 CTTCGCCCAC CCTAGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC

 3061 CCGCGCTATG ACGGCAATAA AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA

 3121 TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG

 3181 GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG

 3241 CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA

 3301 TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT

 3361 TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA

 3421 CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG

 3481 CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC

 3541 AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT

 3601 AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC

 3661 TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT

 3721 GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG

 3781 CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT

 3841 ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG

 3901 GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG

 3961 TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG

 4021 GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG

 4081 GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC

 4141 CGCCATGCAT

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