psiCHECK-2哺乳报告质粒

基本信息

别名:	psiCHECK2，psiCHECK 2
启动子:	SV40，T7
复制子:	pUC ori，SV40 ori
终止子:	SV40 poly(A) signal
质粒分类:	哺乳细胞，信号通路报告载体
质粒大小:	6273bp
原核抗性:	氨苄青霉素Amp
筛选标记:	Luc/hRluc
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:	psiCHECK-2-F：GAAGTTCCCTAACACCGAGT
3'测序引物:	psiCHECK-2-R：CCCCCGCGAGGTCCGAAGAC

质粒简介

        psiCHECK-2 Vector is designed to provide a quantitative and rapid approach for optimization of RNA interference (RNAi). The vectors enable the monitoring of changes in expression of a target gene fused to the reporter gene. In both vectors, Renilla luciferase is used as a primary reporter gene, and the gene of interest can be cloned into the multiple cloning region located downstream of the Renilla luciferase translational stop codon. Initiation of the RNAi process toward a gene of interest results in cleavage and subsequent degradation of fusion mRNA. Measurement of decreased Renilla luciferase activity is a convenient indicator of RNAi effect .
        RNAi is a phenomenon by which double-stranded RNA complementary to a target mRNA can specifically inactivate gene function by stimulating the degradation of the target mRNA . Because of the ability to inactivate genes, RNAi has emerged as a powerful tool for analyzing gene function.
        In mammalian systems, including cultured mammalian cells, chemically synthesized double-stranded short interfering RNA molecules (<30 nucleotides; siRNA) result in dsRNA duplexes <30 base pairs in length that induce RNAi .RNAi duplexes >30bp induce the interferon response and nonspecific degradation of mRNA and cannot be used as tools for specific gene silencing .

质粒图谱

质粒序列

LOCUS       Exported                6273 bp ds-DNA     circular SYN 07-JUL-2016
DEFINITION  synthetic circular DNA
KEYWORDS    psiCHECK-2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6273)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6273
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        62..419
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      270..405
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     intron          489..621
                     /note="chimeric intron"
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        666..684
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             694..1629
                     /codon_start=1
                     /product="Renilla luciferase"
                     /note="hRluc"
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    1688..1736
                     /note="synthetic polyadenylation signal"
     promoter        1745..2496
                     /note="HSV TK promoter"
                     /note="herpes simplex virus thymidine kinase promoter"
     CDS             2532..4184
                     /codon_start=1
                     /note="hluc+"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     polyA_signal    4228..4349
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     promoter        4482..4586
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             4587..5447
                     /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      5618..6206
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
      421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
      481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      661 gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag
      721 caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg
      781 ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt
      841 ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc
      901 gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat
      961 ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac
     1021 cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc ctttcactac
     1081 tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt cgtggacgtg
     1141 atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat caagagcgaa
     1201 gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct cccaagcaag
     1261 atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt caaggagaag
     1321 ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt taagggaggc
     1381 aagcccgacg tcgtccagat tgtccgcaac tacaacgcct accttcgggc cagcgacgat
     1441 ctgcctaaga tgttcatcga gtccgaccct gggttctttt ccaacgctat tgtcgaggga
     1501 gctaagaagt tccctaacac cgagttcgtg aaggtgaagg gcctccactt cagccaggag
     1561 gacgctccag atgaaatggg taagtacatc aagagcttcg tggagcgcgt gctgaagaac
     1621 gagcagtaat tctaggcgat cgctcgagcc cgggaattcg tttaaaccta gagcggccgc
     1681 tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgagga
     1741 tctaaatgag tcttcggacc tcgcgggggc cgcttaagcg gtggttaggg tttgtctgac
     1801 gcggggggag ggggaaggaa cgaaacactc tcattcggag gcggctcggg gtttggtctt
     1861 ggtggccacg ggcacgcaga agagcgccgc gatcctctta agcacccccc cgccctccgt
     1921 ggaggcgggg gtttggtcgg cgggtggtaa ctggcgggcc gctgactcgg gcgggtcgcg
     1981 cgccccagag tgtgaccttt tcggtctgct cgcagacccc cgggcggcgc cgccgcggcg
     2041 gcgacgggct cgctgggtcc taggctccat ggggaccgta tacgtggaca ggctctggag
     2101 catccgcacg actgcggtga tattaccgga gaccttctgc gggacgagcc gggtcacgcg
     2161 gctgacgcgg agcgtccgtt gggcgacaaa caccaggacg gggcacaggt acactatctt
     2221 gtcacccgga ggcgcgaggg actgcaggag cttcagggag tggcgcagct gcttcatccc
     2281 cgtggcccgt tgctcgcgtt tgctggcggt gtccccggaa gaaatatatt tgcatgtctt
     2341 tagttctatg atgacacaaa ccccgcccag cgtcttgtca ttggcgaatt cgaacacgca
     2401 gatgcagtcg gggcggcgcg gtcccaggtc cacttcgcat attaaggtga cgcgtgtggc
     2461 ctcgaacacc gagcgaccct gcagcgaccc gcttaaaagc ttggcattcc ggtactgttg
     2521 gtaaagccac catggccgat gctaagaaca ttaagaaggg ccctgctccc ttctaccctc
     2581 tggaggatgg caccgctggc gagcagctgc acaaggccat gaagaggtat gccctggtgc
     2641 ctggcaccat tgccttcacc gatgcccaca ttgaggtgga catcacctat gccgagtact
     2701 tcgagatgtc tgtgcgcctg gccgaggcca tgaagaggta cggcctgaac accaaccacc
     2761 gcatcgtggt gtgctctgag aactctctgc agttcttcat gccagtgctg ggcgccctgt
     2821 tcatcggagt ggccgtggcc cctgctaacg acatttacaa cgagcgcgag ctgctgaaca
     2881 gcatgggcat ttctcagcct accgtggtgt tcgtgtctaa gaagggcctg cagaagatcc
     2941 tgaacgtgca gaagaagctg cctatcatcc agaagatcat catcatggac tctaagaccg
     3001 actaccaggg cttccagagc atgtacacat tcgtgacatc tcatctgcct cctggcttca
     3061 acgagtacga cttcgtgcca gagtctttcg acagggacaa aaccattgcc ctgatcatga
     3121 acagctctgg gtctaccggc ctgcctaagg gcgtggccct gcctcatcgc accgcctgtg
     3181 tgcgcttctc tcacgcccgc gaccctattt tcggcaacca gatcatcccc gacaccgcta
     3241 ttctgagcgt ggtgccattc caccacggct tcggcatgtt caccaccctg ggctacctga
     3301 tttgcggctt tcgggtggtg ctgatgtacc gcttcgagga ggagctgttc ctgcgcagcc
     3361 tgcaagacta caaaattcag tctgccctgc tggtgccaac cctgttcagc ttcttcgcta
     3421 agagcaccct gatcgacaag tacgacctgt ctaacctgca cgagattgcc tctggcggcg
     3481 ccccactgtc taaggaggtg ggcgaagccg tggccaagcg ctttcatctg ccaggcatcc
     3541 gccagggcta cggcctgacc gagacaacca gcgccattct gattacccca gagggcgacg
     3601 acaagcctgg cgccgtgggc aaggtggtgc cattcttcga ggccaaggtg gtggacctgg
     3661 acaccggcaa gaccctggga gtgaaccagc gcggcgagct gtgtgtgcgc ggccctatga
     3721 ttatgtccgg ctacgtgaat aaccctgagg ccacaaacgc cctgatcgac aaggacggct
     3781 ggctgcactc tggcgacatt gcctactggg acgaggacga gcacttcttc atcgtggacc
     3841 gcctgaagtc tctgatcaag tacaagggct accaggtggc cccagccgag ctggagtcta
     3901 tcctgctgca gcaccctaac attttcgacg ccggagtggc cggcctgccc gacgacgatg
     3961 ccggcgagct gcctgccgcc gtcgtcgtgc tggaacacgg caagaccatg accgagaagg
     4021 agatcgtgga ctatgtggcc agccaggtga caaccgccaa gaagctgcgc ggcggagtgg
     4081 tgttcgtgga cgaggtgccc aagggcctga ccggcaagct ggacgcccgc aagatccgcg
     4141 agatcctgat caaggctaag aaaggcggca agatcgccgt gtaataattc tagagtcggg
     4201 gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa
     4261 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     4321 taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc
     4381 aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
     4441 aaatcgataa ggatccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4501 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4561 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4621 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     4681 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     4741 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     4801 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     4861 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4921 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4981 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5041 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5101 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5161 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5221 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5281 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5341 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5401 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5461 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5521 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5581 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5641 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     5701 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     5761 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5821 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5881 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5941 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6001 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6061 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6121 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6181 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6241 ccttttgctg gccttttgct cacatggctc gac