pACT哺乳双杂交质粒

基本信息

启动子:	CMV promoter
复制子:	pUC ori,f1 ori
终止子:	SV40 poly(A) signal
质粒分类:	哺乳细胞，哺乳细胞双杂交载体
质粒大小:	5566bp
原核抗性:	氨苄青霉素Amp
筛选标记:	遗传霉素G418
克隆菌株:	大肠杆菌DH5α
培养条件:	37℃，有氧 LB
表达宿主:	哺乳细胞
诱导方式:	无须诱导，瞬时表达
5'测序引物:	CMV-F：CGCAAATGGGCGGTAGGCGTG
3'测序引物:	PIRES.R：CACTGCATTCTAGTTGTGGT

质粒简介

          The pACT Vector is a high-copy plasmid in which the human cytomegalovirus (CMV) immediate early promoter drives expression of the herpes virus VP16activation domain (amino acids 411–456). A chimeric intron is located 5´ of the gene segment, and a multiple cloning region is located 3´ of the gene segment for insertion of cDNA clones of interest . The presence of this chimeric intron, in context with the CMV promoter and polyadenylation signal, can result in increased protein expression of cDNA genes linked to these elements. The stop codons and SV40 late polyadenylation region, demonstrated to be efficient for mRNA production , are at the 3´ end of the fusion gene. The fusion gene region is flanked by T7 and T3 RNA polymerase promoters for the synthesis of sense and antisense RNA products. The T7 promoter allows the construct to be translated using the TNT® T7 Quick Coupled Transcription/Translation System (Cat.). Also located on this vector is the neomycin phosphotransferase gene (from Tn5) driven by the SV40 early promoter and followed by a synthetic polyadenylation cassette. This gene confers resistance to the neomycin analog, G418 . The plasmid backbone contains an f1 origin of replication for the production of single-stranded DNA (ssDNA) and the β-lactamase (Ampr) gene for selection of the vector DNA in E. coli.
 

质粒图谱

质粒序列

LOCUS       Exported                5566 bp ds-DNA     circular SYN 23-NOV-2015
DEFINITION  synthetic circular DNA
KEYWORDS    pACT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5566)
  AUTHORS   wang
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5566
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        138..517
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          890..1022
                     /note="chimeric intron"
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        1067..1085
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1116..1148
                     /codon_start=1
                     /note="GAL4 aa1-11"
                     /translation="MKLLSSIEQAC"
     CDS             1149..1172
                     /codon_start=1
                     /note="SV40 large T nuclear targeting sequence"
                     /translation="PKKKRKVD"
     CDS             1149..1169
                     /codon_start=1
                     /product="nuclear localization signal of SV40 large T 
                     antigen"
                     /note="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             1188..1325
                     /codon_start=1
                     /note="VP16 fusion protein"
                     /translation="STAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGP
                     G"
     misc_feature    1334..1382
                     /note="MCS"
     promoter        complement(1403..1420)
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    1438..1559
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      1693..2148
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2163..2520
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2371..2506
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2571..3365
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3429..3477
                     /note="synthetic polyadenylation signal"
     promoter        3769..3873
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             3874..4734
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4905..5493
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc cagcttgaag caagcctcct gaaagatgaa gctactgtct tctatcgaac
     1141 aagcatgccc aaaaaagaag agaaaggtag atgaattccc ggggatctcg acggcccccc
     1201 cgaccgatgt cagcctgggg gacgagctcc acttagacgg cgaggacgtg gcgatggcgc
     1261 atgccgacgc gctagacgat ttcgatctgg acatgttggg ggacggggat tccccgggtc
     1321 cgggatcgcc agggatccgt cgacttgacg cgttgatatc atctagagcg gccgcaggta
     1381 cctgaataac taaggccgct tccctttagt gagggttaat gcttcgagca gacatgataa
     1441 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     1501 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     1561 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     1621 aaagcaagta aaacctctac aaatgtggta aaatccgata aggatcgatt ccggagcctg
     1681 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1741 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1801 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1861 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1921 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1981 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2041 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2101 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcg cctgtgtacc
     2161 ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2221 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2281 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     2341 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     2401 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     2461 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     2521 aagcttgatt cttctgacac aacagtctcg aacttaaggc tagagccacc atgattgaac
     2581 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     2641 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     2701 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     2761 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     2821 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     2881 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     2941 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     3001 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     3061 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     3121 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     3181 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     3241 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     3301 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     3361 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     3421 atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaag
     3481 atccgcgtat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     3541 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3601 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3661 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     3721 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     3781 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3841 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3901 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3961 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4021 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     4081 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     4141 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     4201 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     4261 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     4321 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     4381 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     4441 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     4501 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     4561 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     4621 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     4681 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     4741 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4801 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4861 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4921 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4981 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5041 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5101 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5161 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5221 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5281 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5341 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5401 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5461 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5521 ttcctggcct tttgctggcc ttttgctcac atggctcgac agatct