pAmCyan1-C1

pAmCyan1-C1

pAmCyan1-C1载体基本信息

pAmCyan1-C1载体质粒图谱和多克隆位点信息

pAmCyan1-C1载体描述

pAmCyan1-C1 encodes a human codon-optimized variant of wild-type Anemonia majano cyan fluorescent protein, AmCyan1 (1). The AmCyan1 coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for optimal expression in mammalian cells (2). Additionally, an upstream sequence—located just 5' to the AmCyan1 start codon—has been converted to a Kozak consensus translation initiation site (3) to further increase the translation efficiency in eukaryotic cells. Two amino acid substitutions (Asn-34 to Ser; Lys-68 to Met) have been made to enhance the emission characteristics of AmCyan1 (excitation maximum = 458 nm; emission maximum = 489 nm).

The multiple cloning site (MCS) in pAmCyan1-C1 is positioned between the AmCyan1 coding sequence and a pair of SV40 polyadenylation signals (SV40 poly A). Thus, genes cloned into the MCS will be expressed as fusions to the C-terminus of AmCyan1 if they are in the same reading frame as AmCyan1 and there are no intervening stop codons. Expression of AmCyan1 is driven by the cytomegalovirus immediate-early promoter (PCMV IE). The SV40 poly A signals downstream of the MCS direct proper processing of the 3' end of AmCyan1 mRNA. The vector backbone contains an SV40 origin (SV40 ori) for replication in mammalian cells that express 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—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK poly A) gene—allows stably transfected eukaryotic cells to be selected using G418 (4). A bacterial promoter (P) upstream of this cassette drives expression of the Neor/Kanr gene in E. coli hosts, which can be selected with kanamycin.Fusions to the C terminus of AmCyan1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo. The target gene should be cloned into pAmCyan1-C1 so that it is in frame with the AmCyan1 coding sequence, with no intervening, in-frame stop codons. The recombinant pAmCyan1-C1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418. We recommend selecting mammalian cell cultures in 500–1,300 μg/ml G418, depending on the cell line. Be sure to establish a kill curve for each cell line and each lot of G418 to determine the optimal selection concentration. Unmodified (i.e., non-recombinant) pAmCyan1-C1 can also be used simply to express AmCyan1 in a cell line of interest (e.g., as a transfection marker).

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) to E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ~500

 Plasmid incompatibility group: pMB1/Col E1

pAmCyan1-C1载体序列

ORIGIN

    1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG

   61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT

  121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

  181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC

  241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

  301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC

  361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG

  421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

  481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT

  541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA

  601 CCGGTCGCCA CCATGGCCCT GTCCAACAAG TTCATCGGCG ACGACATGAA GATGACCTAC

  661 CACATGGACG GCTGCGTGAA CGGCCACTAC TTCACCGTGA AGGGCGAGGG CAGCGGCAAG

  721 CCCTACGAGG GCACCCAGAC CTCCACCTTC AAGGTGACCA TGGCCAACGG CGGCCCCCTG

  781 GCCTTCTCCT TCGACATCCT GTCCACCGTG TTCATGTACG GCAACCGCTG CTTCACCGCC

  841 TACCCCACCA GCATGCCCGA CTACTTCAAG CAGGCCTTCC CCGACGGCAT GTCCTACGAG

  901 AGAACCTTCA CCTACGAGGA CGGCGGCGTG GCCACCGCCA GCTGGGAGAT CAGCCTGAAG

  961 GGCAACTGCT TCGAGCACAA GTCCACCTTC CACGGCGTGA ACTTCCCCGC CGACGGCCCC

 1021 GTGATGGCCA AGAAGACCAC CGGCTGGGAC CCCTCCTTCG AGAAGATGAC CGTGTGCGAC

 1081 GGCATCTTGA AGGGCGACGT GACCGCCTTC CTGATGCTGC AGGGCGGCGG CAACTACAGA

 1141 TGCCAGTTCC ACACCTCCTA CAAGACCAAG AAGCCCGTGA CCATGCCCCC CAACCACGTG

 1201 GTGGAGCACC GCATCGCCAG AACCGACCTG GACAAGGGCG GCAACAGCGT GCAGCTGACC

 1261 GAGCACGCCG TGGCCCACAT CACCTCCGTG GTGCCCTTCT CCGGACTCAG ATCTCGAGCT

 1321 CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG GATCCACCGG ATCTAGATAA

 1381 CTGATCATAA TCAGCCATAC CACATTTGTA GAGGTTTTAC TTGCTTTAAA AAACCTCCCA

 1441 CACCTCCCCC TGAACCTGAA ACATAAAATG AATGCAATTG TTGTTGTTAA CTTGTTTATT

 1501 GCAGCTTATA ATGGTTACAA ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT

 1561 TTTTCACTGC ATTCTAGTTG TGGTTTGTCC AAACTCATCA ATGTATCTTA ACGCGTAAAT

 1621 TGTAAGCGTT AATATTTTGT TAAAATTCGC GTTAAATTTT TGTTAAATCA GCTCATTTTT

 1681 TAACCAATAG GCCGAAATCG GCAAAATCCC TTATAAATCA AAAGAATAGA CCGAGATAGG

 1741 GTTGAGTGTT GTTCCAGTTT GGAACAAGAG TCCACTATTA AAGAACGTGG ACTCCAACGT

 1801 CAAAGGGCGA AAAACCGTCT ATCAGGGCGA TGGCCCACTA CGTGAACCAT CACCCTAATC

 1861 AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC ACTAAATCGG AACCCTAAAG GGAGCCCCCG

 1921 ATTTAGAGCT TGACGGGGAA AGCCGGCGAA CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA

 1981 AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT AGCGGTCACG CTGCGCGTAA CCACCACACC

 2041 CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG

 2101 CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA

 2161 ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG AGTCCTGAGG CGGAAAGAAC

 2221 CAGCTGTGGA ATGTGTGTCA GTTAGGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA

 2281 AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC

 2341 CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC

 2401 CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC

 2461 TGACTAATTT TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CGGCCTCTGA GCTATTCCAG

 2521 AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAGATCGATC AAGAGACAGG

 2581 ATGAGGATCG TTTCGCATGA TTGAACAAGA TGGATTGCAC GCAGGTTCTC CGGCCGCTTG

 2641 GGTGGAGAGG CTATTCGGCT ATGACTGGGC ACAACAGACA ATCGGCTGCT CTGATGCCGC

 2701 CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC GGTTCTTTTT GTCAAGACCG ACCTGTCCGG

 2761 TGCCCTGAAT GAACTGCAAG ACGAGGCAGC GCGGCTATCG TGGCTGGCCA CGACGGGCGT

 2821 TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC TGAAGCGGGA AGGGACTGGC TGCTATTGGG

 2881 CGAAGTGCCG GGGCAGGATC TCCTGTCATC TCACCTTGCT CCTGCCGAGA AAGTATCCAT

 2941 CATGGCTGAT GCAATGCGGC GGCTGCATAC GCTTGATCCG GCTACCTGCC CATTCGACCA

 3001 CCAAGCGAAA CATCGCATCG AGCGAGCACG TACTCGGATG GAAGCCGGTC TTGTCGATCA

 3061 GGATGATCTG GACGAAGAGC ATCAGGGGCT CGCGCCAGCC GAACTGTTCG CCAGGCTCAA

 3121 GGCGAGCATG CCCGACGGCG AGGATCTCGT CGTGACCCAT GGCGATGCCT GCTTGCCGAA

 3181 TATCATGGTG GAAAATGGCC GCTTTTCTGG ATTCATCGAC TGTGGCCGGC TGGGTGTGGC

 3241 GGACCGCTAT CAGGACATAG CGTTGGCTAC CCGTGATATT GCTGAAGAGC TTGGCGGCGA

 3301 ATGGGCTGAC CGCTTCCTCG TGCTTTACGG TATCGCCGCT CCCGATTCGC AGCGCATCGC

 3361 CTTCTATCGC CTTCTTGACG AGTTCTTCTG AGCGGGACTC TGGGGTTCGA AATGACCGAC

 3421 CAAGCGACGC CCAACCTGCC ATCACGAGAT TTCGATTCCA CCGCCGCCTT CTATGAAAGG

 3481 TTGGGCTTCG GAATCGTTTT CCGGGACGCC GGCTGGATGA TCCTCCAGCG CGGGGATCTC

 3541 ATGCTGGAGT TCTTCGCCCA CCCTAGGGGG AGGCTAACTG AAACACGGAA GGAGACAATA

 3601 CCGGAAGGAA CCCGCGCTAT GACGGCAATA AAAAGACAGA ATAAAACGCA CGGTGTTGGG

 3661 TCGTTTGTTC ATAAACGCGG GGTTCGGTCC CAGGGCTGGC ACTCTGTCGA TACCCCACCG

 3721 AGACCCCATT GGGGCCAATA CGCCCGCGTT TCTTCCTTTT CCCCACCCCA CCCCCCAAGT

 3781 TCGGGTGAAG GCCCAGGGCT CGCAGCCAAC GTCGGGGCGG CAGGCCCTGC CATAGCCTCA

 3841 GGTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT TTTAATTTAA AAGGATCTAG

 3901 GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC

 3961 TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC

 4021 GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT

 4081 CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT

 4141 ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT

 4201 ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT

 4261 CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG

 4321 GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA

 4381 CAGCGTGAGC TATGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG

 4441 GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG

 4501 TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC

 4561 TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG

 4621 GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGAT

 4681 AACCGTATTA CCGCCATGCA T

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