pAmCyan

pAmCyan

pAmCyan载体基本信息

pAmCyan载体质粒图谱和多克隆位点信息

pAmCyan载体简介

pAmCyan is a pUC19-derived prokaryotic expression vector, which encodes a variant of wildtype Anemonia majano cyan fluorescent protein (AmCyan; 1) that has been engineered for brighter fluorescence. Two amino acid substitutions (Asn-34 to Ser; Lys-68 to Met) have been made to enhance the emission characteristics of AmCyan (excitation maximum = 458 nm;emission maximum = 489 nm).

The AmCyan gene was inserted in frame with the lacZ initiation codon from pUC19 so that AmCyan is expressed from the lac promoter (Plac) in E. coli. The AmCyan coding sequence is flanked by distinct multiple cloning sites (MCS) at the 5' and 3' ends so that the gene can be readily excised from pAmCyan and subcloned into other expression vectors. An upstream sequence—located just 5' to the AmCyan gene—has been converted to a Kozak consensus translation initiation site (2) to increase the translation efficiency in eukaryotic expression systems. The pUC backbone of pAmCyan provides a high-copy-number origin of replication (pUC ori) and an ampicillin resistance gene (Ampr) for propagation and selection in E. coli.

pAmCyan Vector serves as a convenient source of AmCyan cDNA. The flanking MCS regions make it possible to excise the AmCyan coding sequence and insert it into other expression vectors. Alternatively, the AmCyan coding sequence can be amplified by PCR.

Propagation in E. coli

Þ        Recommended host strain: JM109

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

Þ        E. coli replication origin: pUC

Þ        Copy number: ~500

Þ        Plasmid incompatibility group: pMB1/Col E1

载体序列

ORIGIN

    1 AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC

   61 ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTGAGTTAGC

  121 TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA

  181 TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCTTG

  241 CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGG TCGCCACCAT GGCTCTTTCA

  301 AACAAGTTTA TCGGAGATGA CATGAAAATG ACCTACCATA TGGATGGCTG TGTCAATGGG

  361 CATTACTTTA CCGTCAAAGG TGAAGGCAGC GGGAAGCCAT ACGAAGGGAC GCAGACCTCG

  421 ACTTTTAAAG TCACCATGGC CAACGGTGGG CCCCTTGCAT TCTCCTTTGA CATACTATCT

  481 ACAGTGTTCA TGTATGGAAA TCGATGCTTT ACTGCGTATC CTACCAGTAT GCCCGACTAT

  541 TTCAAACAAG CATTTCCTGA CGGAATGTCA TATGAAAGGA CTTTTACCTA TGAAGATGGA

  601 GGAGTTGCTA CAGCCAGTTG GGAAATAAGC CTTAAAGGCA ACTGCTTTGA GCACAAATCC

  661 ACGTTTCATG GAGTGAACTT TCCTGCTGAT GGACCTGTGA TGGCGAAGAT GACAACTGGT

  721 TGGGACCCAT CTTTTGAGAA AATGACTGTC TGCGATGGAA TATTGAAGGG TGATGTCACC

  781 GCGTTCCTCA TGCTGCAAGG AGGTGGCAAT TACAGATGCC AATTCCACAC TTCTTACAAG

  841 ACAAAAAAAC CGGTGACGAT GCCACCAAAC CATGCGGTGG AACATCGCAT TGCGAGGACC

  901 GACCTTGACA AAGGTGGCAA CAGTGTTCAG CTGACGGAGC ACGCTGTTGC ACATATAACC

  961 TCTGTTGTCC CTTTCTGAAG CGGCCGCGAC TCTAGAATTC CAACTGAGCG CCGGTCGCTA

 1021 CCATTACCAA CTTGTCTGGT GTCAAAAATA ATAGGCCTAC TAGTCGGCCG TACGGGCCCT

 1081 TTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG CTCCCGGAGA

 1141 CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG GGCGCGTCAG

 1201 CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA

 1261 GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAAAA TACCGCATCA

 1321 GGCGGCCTTA AGGGCCTCGT GATACGCCTA TTTTTATAGG TTAATGTCAT GATAATAATG

 1381 GTTTCTTAGA CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA

 1441 TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT

 1501 CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC CCTTATTCCC

 1561 TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT GAAAGTAAAA

 1621 GATGCTGAAG ATCAGTTGGG TGCACGAGTG GGTTACATCG AACTGGATCT CAACAGCGGT

 1681 AAGATCCTTG AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT

 1741 CTGCTATGTG GCGCGGTATT ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGC

 1801 ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG

 1861 GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG

 1921 GCCAACTTAC TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT TTTGCACAAC

 1981 ATGGGGGATC ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA

 2041 AACGACGAGC GTGACACCAC GATGCCTGTA GCAATGGCAA CAACGTTGCG CAAACTATTA

 2101 ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT GGAGGCGGAT

 2161 AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA

 2221 TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC AGATGGTAAG

 2281 CCCTCCCGTA TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT

 2341 AGACAGATCG CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC AGACCAAGTT

 2401 TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG

 2461 AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGA

 2521 GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA

 2581 ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA

 2641 GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT

 2701 GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA

 2761 TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT

 2821 ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG

 2881 GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG

 2941 CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA

 3001 AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT

 3061 CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG

 3121 TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC

 3181 TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC

 3241 CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC

 3301 GAGTCAGTGA GCGAGGAAGC GGAAG

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