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載體描述

DsRed-Express is a human codon-optimized DsRed variant of Discosoma sp. red fluorescent protein. Its coding sequence contains nine amino acid substitutions which improve solubility of the protein and reduce the time from transfection to detection of red fluorescence (exitation and emission maxima = 557 nm and 579 nm, respectively). In addition, these substitutions reduce the level of residual green emission. Although DsRed-Express most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express displays a reduced tendency to aggregate.

The MCS in pIRES2-DsRed-Express is between the immediate early promoter of cytomegalovirus (P CMV IE ) and the IRES sequence. SV40 polyadenylation signals downstream of the DsRed-Express gene direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neo r ), consisting 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pIRES2-DsRed-Express backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體序列

LOCUS       Exported                5264 bp ds-DNA     circular SYN 20-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 5264)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Wednesday, Mar 20, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..5264

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        61..364

                     /label=CMV enhancer

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        365..568

                     /label=CMV promoter

                     /note="human cytomegalovirus (CMV) immediate early

                     promoter"

     misc_feature    591..665

                     /label=MCS

                     /note="multiple cloning site"

     misc_feature    667..1253

                     /label=IRES2

                     /note="internal ribosome entry site (IRES) of the

                     encephalomyocarditis virus (EMCV)"

     CDS             1254..1931

                     /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

                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK

                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH

                     LFL"

     polyA_signal    2052..2173

                     /label=SV40 poly(A) signal

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(2180..2635)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     promoter        2662..2766

                     /gene="bla"

                     /label=AmpR promoter

     promoter        2768..3125

                     /label=SV40 promoter

                     /note="SV40 enhancer and early promoter"

     rep_origin      2976..3111

                     /label=SV40 ori

                     /note="SV40 origin of replication"

     CDS             3160..3954

                     /codon_start=1

                     /gene="aph(3')-II (or nptII)"

                     /product="aminoglycoside phosphotransferase from Tn5"

                     /label=NeoR/KanR

                     /note="confers resistance to neomycin, kanamycin, and G418

                     (Geneticin(R))"

                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP

                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS

                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    4186..4233

                     /label=HSV TK poly(A) signal

                     /note="herpes simplex virus thymidine kinase

                     polyadenylation signal (Cole and Stacy, 1985)"

     rep_origin      4562..5150

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg

      661 gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg

      721 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag

      781 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc

      841 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg

      901 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag

      961 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca

     1021 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt

     1081 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc

     1141 tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa

     1201 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca accatggcct

     1261 cctccgagga cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga

     1321 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga

     1381 ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc

     1441 cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca

     1501 agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg

     1561 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg ctccttcatc tacaaggtga

     1621 agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct

     1681 gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca

     1741 aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtct atctatatgg

     1801 ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct

     1861 cccacaacga ggactacacc atcgtggagc agtacgagcg cgccgagggc cgccaccacc

     1921 tgttcctgta gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt

     1981 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa

     2041 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca

     2101 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca

     2161 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat

     2221 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa

     2281 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta

     2341 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca

     2401 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat

     2461 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg

     2521 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc

     2581 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt

     2641 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca

     2701 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg

     2761 aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt

     2821 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca

     2881 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt

     2941 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt

     3001 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg

     3061 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt

     3121 gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg

     3181 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag

     3241 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt

     3301 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta

     3361 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg

     3421 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt

     3481 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat

     3541 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg

     3601 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca

     3661 gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc

     3721 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc

     3781 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat

     3841 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc

     3901 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga

     3961 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt

     4021 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga

     4081 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa

     4141 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac

     4201 agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct

     4261 ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct

     4321 tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg

     4381 cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc

     4441 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc

     4501 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt

     4561 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac

     4621 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct

     4681 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact

     4741 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg

     4801 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata

     4861 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga

     4921 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag

     4981 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg

     5041 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac

     5101 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca

     5161 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg

     5221 cgttatcccc tgattctgtg gataaccgta ttaccgccat gcat

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